Microfluidic free interface diffusion techniques

ABSTRACT

A static fluid and a second fluid are placed into contact along a microfluidic free interface and allowed to mix by diffusion without convective flow across the interface. In accordance with one embodiment of the present invention, the fluids are static and initially positioned on either side of a closed valve structure in a microfluidic channel having a width that is tightly constrained in at least one dimension. The valve is then opened, and no-slip layers at the sides of the microfluidic channel suppress convective mixing between the two fluids along the resulting interface. Applications for microfluidic free interfaces in accordance with embodiments of the present invention include, but are not limited to, protein crystallization studies, protein solubility studies, determination of properties of fluidics systems, and a variety of biological assays such as diffusive immunoassays, substrate turnover assays, and competitive binding assays.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/213,990, filed Aug. 19, 2011, which is a continuation of U.S. patentapplication Ser. No. 12/762,170, filed Apr. 16, 2010, which is adivisional of U.S. patent application Ser. No. 12/001,768, filed Dec.11, 2007, issued as U.S. Pat. No. 7,704,322 on Apr. 27, 2010, which is adivisional of U.S. patent application Ser. No. 10/265,473, filed Oct. 4,2002, issued as U.S. Pat. No. 7,306,672 on Dec. 11, 2007, which is acontinuation-in-part of U.S. patent application Ser. No. 09/826,583,filed Apr. 6, 2001, issued as U.S. Pat. No. 6,899,137 on May 31, 2005,and is a continuation-in-part of U.S. patent application Ser. No.09/887,997, filed Jun. 22, 2001, issued as U.S. Pat. No. 7,052,545 onMay 30, 2006, and is a continuation-in-part of U.S. patent applicationSer. No. 10/117,978, filed Apr. 5, 2002, now issued as U.S. Pat. No.7,195,670 on Mar. 27, 2007. These prior patent applications are herebyincorporated by reference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.HG-01642-02 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Free-interface diffusion techniques have been employed in a number ofscientific applications, most notably in protein crystallizationstudies. In a free interface, mixing between different fluids occursentirely as a result of diffusion. The creation of such free interfacesoffer the advantage of gradual and non-directional mixing, avoidingasymmetries and steep concentration gradients associated with convectiveflow between the fluids.

However, certain technical difficulties have rendered conventionalmacroscopic free interfaces unsuitable for high throughput screeningapplications, and they are not currently widely used in thecrystallographic community for several reasons.

First, conventional macroscopic fluidic interfaces are established bydispensing solutions into a narrow container; such as a capillary tubehaving a width of 100 μm or greater, or a deep well in a culture plate.FIGS. 30A-B show simplified cross-sectional views of the attemptedformation of a macroscopic free-interface in a capillary tube 9300. Theact of dispensing a second fluid 9302 into a first fluid 9304 may giverise to convective mixing and result in a poorly defined fluidicinterface 9306.

Unfortunately, the fluids may not be sucked into a capillary serially toeliminate this problem. FIGS. 31A-B show the mixing, between a firstsolution 9400 and a second solution 9402 in a capillary tube 9404 thatwould result due to the parabolic velocity distribution of pressuredriven convective Poiseuille flow, also resulting in a poorly definedfluidic interface 9406.

Moreover, the container for a macroscopic free-interface crystallizationregime must have dimensions making them accessible to a pipette tip,needle, capillary or other g tool, and necessitating the use ofrelatively large (10-100 μl) fluid volumes.

In order to avoid unwanted convective mixing during macroscopic freeinterface diffusion experiments, considerable care must be exercisedboth during dispensing of the fluids and during the diffusion period.For this reason cumbersome protocols are often used to define amacroscopic free-interface. In one conventional approach, one fluid maybe frozen or otherwise converted to solid phase prior to addition of thesecond fluid. In an alternative conventional approach, the one fluidcontaining the sample is converted into a solid through polymerization,trapping the sample within the polymer, which is then exposed to thesecond fluid. See generally Garcia-Ruiz et al., “Agarose asCrystallization Media for Proteins I: Transport Processes”, J. CrystalGrowth 232, 165-172 (2001).

The problems of convective mixing associated with macroscopic freeinterface diffusion studies is compounded in that two fluids ofdiffering density will mix by gravity induced convection if they are notstored at the proper orientation, additionally complicating the storageof reactions. This is shown in FIGS. 32A-C, wherein over time firstsolution 9500 having a density greater than the density of secondsolution 9502 merely sinks to form a static bottom layer 9504 that isnot conducive to formation of a diffusion gradient along the length of acapillary tube.

Other approaches to the formation of free interfaces have focused uponmicrofluidic structures. For example, Kamholz et al., “QuantitativeAnalysis of Molecular Interaction in a Microfluidic Channel: theT-Sensor”, Anal. Chem. Vol. 71, No. 23 (1999), describe the formation ofa diffusive interface between two flowing fluids inlet on sides ofmicrofluidic T-shaped junction and then moving together along the stem.The dimensions of the microfluidic channels impose laminar flow on thefluids, such that convective mixing between them is eliminated anddiffusion only occurs across the interface.

While the diffusion described by Kamholz et al. may be useful, it offersthe distinct disadvantage of necessarily creating an elongated interfacebetween the fluids, such that substantial volumes of the fluids arecontinuously exposed to the steep concentration gradient occurring atthe interface between them. Such a steep gradient poses a number ofdisadvantages. In the context of a protein crystallization experimentalprotocol, one such disadvantage is the possibility of solvent shock andthe precipitation of sample in non-crystalline form along the interface.

Accordingly, there is a need in the art for methods and structures foraccomplishing diffusive introduction of material under highly controlledconditions that limit the exposure of volumes of solution to steepconcentration gradients.

BRIEF SUMMARY OF THE INVENTION

A static fluid and a second fluid are placed into contact along amicrofluidic free interface and allowed to mix by diffusion withoutconvective flow across the interface. In accordance with one embodimentof the present invention, the fluids are static and initially positionedon either side of a closed valve structure in a microfluidic channelhaving a width that is tightly constrained in at least one dimension.The valve is then opened, and no-slip layers at the sides of themicrofluidic channel suppress convective mixing between the two fluidsalong the resulting interface. Applications for microfluidic freeinterfaces in accordance with embodiments of the present inventioninclude, but are not limited to, protein crystallization studies,protein solubility studies, determination of properties of fluidicssystems, and a variety of biological assays such as diffusiveimmunoassays, substrate turnover assays, and competitive binding assays.

An embodiment of a method of mixing two fluids in accordance with thepresent invention comprises disposing a first static fluid, disposing asecond fluid proximate to the first static fluid to form a fluidicinterface, and suppressing convective flow of the first and secondfluids such that mixing between the first and second fluids across theinterface occurs substantially exclusively by diffusion.

An alternative embodiment of a method of mixing two fluids in accordancewith the present invention comprises providing a first static fluidhaving a total volume, providing a second static fluid having a totalvolume, disposing a portion of the first static fluid in contact with aportion of the second static fluid to form a fluidic interface, suchthat a minimum volume of the first and second fluids is exposed to asteepest concentration gradient present immediately along the fluidicinterface.

An embodiment of a method of determining a property of a fluidic systemin accordance with the present invention comprises disposing a firststatic fluid containing a target, disposing a second static fluidproximate to the first fluid to form a fluidic interface, andsuppressing convective flow of the first and second fluids such thatmixing occurs across the interface solely by diffusion to reveal thephysical property.

An embodiment of a structure for analyzing a property of a targetmaterial comprises a first microfabricated region configured to containa volume of a first static fluid including a target, a secondmicrofabricated region configured to contain a volume of a second staticfluid including an analyte known to bind to the target, and a valveactuable to place the volume of the first fluid in diffusivecommunication with the volume of the second fluid across a freemicrofluidic interface. A detector is configured to detect a presence ofthe analyte in the first microfabricated region.

An embodiment of a method of reducing a concentration of a smallmolecule in a protein sample in accordance with the present inventioncomprises disposing a first static fluid containing the protein samplein a microfluidic channel, disposing a second static fluid having a lowconcentration of the small molecule proximate to the first fluid to forma fluidic interface, and suppressing convective flow of the first andsecond fluids such that the small molecule from the first static fluiddiffuses across the interface solely by diffusion to reduce theconcentration of small molecule present in the first static fluid.

An embodiment of a method for determining reaction between a ligand anda target comprises positioning a first fluid containing the ligand in afirst chamber, positioning a second fluid containing the target in asecond chamber, and establishing a microfluidic free interface betweenthe first and second fluids in a channel connecting the first and thesecond chamber. Mixing is allowed to occur by diffusion across themicrofluidic free interface between the first and second fluids, suchthat the ligand binds to the target and reactivity between the ligandand target can be determined by deviation of at least one of a temporaldiffusion profile and a spatial diffusion profile from a correspondingprofile expected in the absence of the target.

An embodiment of a method of sampling a chemical reaction under aspectrum of conditions in accordance with the present inventioncomprises forming a microfluidic free interface between a first fluidcontaining a first reactant and a second fluid containing a secondreactant, and causing diffusion of the first reactant into the secondfluid to create a concentration gradient of the first reactant. Reactionbetween the first reactant at various concentrations along the gradientand the second reactant is observed.

An embodiment of a method of creating a concentration gradient of achemical species in accordance with the present invention comprisesdisposing a first fluid containing the chemical species, and disposing asecond static fluid proximate to the first static fluid to form amicrofluidic free interface. Convective flow of the first and secondfluids is suppressed such that mixing between the first and secondfluids across the microfluidic free interface occurs substantiallyexclusively by diffusion and a concentration gradient of the chemicalspecies is created.

An embodiment of a method of introducing a drug into a subject inaccordance with the present invention comprises disposing a first staticfluid containing the drug in a microfluidic device, and disposing asecond fluid in the microfluidic device between the first static fluidand the subject. A microfluidic free interface is established betweenthe first fluid and the second fluid, such that a predetermined amountof the drug diffuses to reach the subject only after a predeterminedtime.

An embodiment of a method of controlling the concentration of reactantsduring a chemical reaction in accordance with the present inventioncomprises disposing a first static fluid containing a first reactant ina microfluidic structure, and disposing a second fluid in themicrofluidic structure proximate to the first static fluid, the secondstatic fluid containing a second reactant. A microfluidic free interfaceis established between the first and second fluids, and diffusionbetween the first and second fluids across the microfluidic freeinterface is allowed to determine the relative concentration of thefirst and second reactants.

These and other embodiments of the present invention, as well as itsadvantages and features, are described in more detail in conjunctionwith the text below and attached figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a first elastomeric layer formed on top ofa micromachined mold.

FIG. 2 is an illustration of a second elastomeric layer formed on top ofa micromachined mold.

FIG. 3 is an illustration of the elastomeric layer of FIG. 2 removedfrom the micromachined mold and positioned over the top of theelastomeric layer of FIG. 1.

FIG. 4 is an illustration corresponding to FIG. 3, but showing thesecond elastomeric layer positioned on top of the first elastomericlayer.

FIG. 5 is an illustration corresponding to FIG. 4, but showing the firstand second elastomeric layers bonded together.

FIG. 6 is an illustration corresponding to FIG. 5, but showing the firstmicromachined mold removed and a planar substrate positioned in itsplace.

FIG. 7A is an illustration corresponding to FIG. 6, but showing theelastomeric structure sealed onto the planar substrate.

FIG. 7B is a front sectional view corresponding to FIG. 7A, showing anopen flow channel.

FIGS. 7C-7G are illustrations showing steps of a method for forming anelastomeric structure having a membrane formed from a separateelastomeric layer.

FIG. 7H is a front sectional view showing the valve of FIG. 7B in anactuated state.

FIGS. 8A and 8B illustrates valve opening vs. applied pressure forvarious flow channels.

FIG. 9 illustrates time response of a 100 μm×100 μm×10 μm RTVmicrovalve.

FIG. 10 is a front sectional view of the valve of FIG. 7B showingactuation of the membrane.

FIG. 11 is a front sectional view of an alternative embodiment of avalve having a flow channel with a curved upper surface.

FIG. 12A is a top schematic view of an on/off valve.

FIG. 12B is a sectional elevation view along line 23B-23B in FIG. 12A

FIG. 13A is a top schematic view of a peristaltic pumping system.

FIG. 13B is a sectional elevation view along line 24B-24B in FIG. 13A

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for an embodiment of the peristaltic pumping system of FIG.13.

FIG. 15A is a top schematic view of one control line actuating multipleflow lines simultaneously.

FIG. 15B is a sectional elevation view along line 26B-26B in FIG. 15A.

FIG. 16 is a schematic illustration of a multiplexed system adapted topermit flow through various channels.

FIG. 17A is a plan view of a flow layer of an addressable reactionchamber structure.

FIG. 17B is a bottom plan view of a control channel layer of anaddressable reaction chamber structure.

FIG. 17C is an exploded perspective view of the addressable reactionchamber structure formed by bonding the control channel layer of FIG.17B to the top of the flow layer of FIG. 17A.

FIG. 17D is a sectional elevation view corresponding to FIG. 17C, takenalong line 28D-28D in FIG. 17C.

FIG. 18 is a schematic of a system adapted to selectively direct fluidflow into any of an array of reaction wells.

FIG. 19 is a schematic of a system adapted for selectable lateral flowbetween parallel flow channels.

FIG. 20A is a bottom plan view of first layer (i.e.: the flow channellayer) of elastomer of a switchable flow array.

FIG. 20B is a bottom plan view of a control channel layer of aswitchable flow array.

FIG. 20C shows the alignment of the first layer of elastomer of FIG. 20Awith one set of control channels in the second layer of elastomer ofFIG. 20B.

FIG. 20D also shows the alignment of the first layer of elastomer ofFIG. 20A with the other set of control channels in the second layer ofelastomer of FIG. 20B.

FIGS. 21A-J show views of one embodiment of a normally-closed valvestructure in accordance with the present invention.

FIGS. 22A and 22B show plan views illustrating operation of oneembodiment of a side-actuated valve structure in accordance with thepresent invention.

FIG. 23 shows a cross-sectional view of one embodiment of a compositestructure in accordance with the present invention.

FIG. 24 shows a cross-sectional view of another embodiment of acomposite structure in accordance with the present invention.

FIG. 25 shows a cross-sectional view of another embodiment of acomposite structure in accordance with the present invention.

FIGS. 26A-D show plan views illustrating operation of one embodiment ofa cell pen structure in accordance with the present invention.

FIGS. 27A-B show plan and cross-sectional views illustrating operationof one embodiment of a cell cage structure in accordance with thepresent invention.

FIGS. 28A-B show plan views of operation of a wiring structure utilizingcross-channel injection in accordance with the embodiment of the presentinvention.

FIGS. 29A-D illustrate cross-sectional views of metering by volumeexclusion in accordance with an embodiment of the present invention.

FIGS. 30A-B show simplified cross-sectional views of the attemptedformation of a macroscopic free-interface in a capillary tube.

FIGS. 31A-B show simplified cross-sectional views of convective mixingbetween a first solution and a second solution in a capillary tuberesulting from a parabolic velocity distribution of pressure drivenPoiseuille flow.

FIGS. 32A-C show simplified cross-sectional views of interaction in acapillary tube between a first solution having a density greater thanthe density of second solution.

FIG. 33A shows a simplified cross-sectional view of a microfluidic freeinterface in accordance with an embodiment of the present invention.

FIG. 33B shows a simplified cross-sectional view of a conventionalnon-microfluidic interface.

FIGS. 34A-D show plan views of the priming of a flow channel andformation of a microfluidic free interface in accordance with anembodiment of the present invention.

FIGS. 35A-E show simplified schematic views of the use of“break-through” valves to create a microfluidic free interface.

FIGS. 36A-D are simplified schematic diagrams plotting concentrationversus distance for two fluids in diffusing across a microfluidic freeinterface in accordance with an embodiment of the present invention.

FIG. 37A shows a simplified plan view of a flow channel overlapped atintervals by a forked control channel to define a plurality of chambers(A-G) positioned on either side of a separately-actuated interfacevalve.

FIGS. 37B-D plot solvent concentration at different times for the flowchannel shown in FIG. 37A.

FIG. 38A shows three sets of pairs of chambers connected bymicrochannels of a different length.

FIG. 38B plots equilibration time versus channel length.

FIG. 39 shows four pairs of chambers, each having different arrangementsof connecting microchannel(s).

FIG. 40A shows a plan view of a simple embodiment of a microfluidicstructure in accordance with the present invention.

FIG. 40B is a simplified plot of concentration versus distance for thestructure of FIG. 40A.

FIG. 41 plots the time required for the concentration in one of thereservoirs of FIG. 40A to reach 0.6 of the final equilibrationconcentration, versus channel length.

FIG. 42 plots the inverse of the time required for the concentration inone of the reservoirs to reach 0.6 of the final equilibrationconcentration (T_(0.6)), versus the area of the fluidic interface ofFIG. 40A.

FIG. 43 presents a phase diagram depicting the phase space betweenfluids A and B, and the path in phase space traversed in the reservoirsas the fluids diffuse across the microfluidic free interface of FIG.40A.

FIG. 44A shows a simplified schematic view of a protein crystal beingformed utilizing a conventional macroscopic free interface diffusiontechnique.

FIG. 44B shows a simplified schematic view of a protein crystal beingformed utilizing diffusion across a microfluidic free interface inaccordance with an embodiment of the present invention.

FIG. 45A shows a simplified plan view of one embodiment of amicrofluidic structure for performing diffusion analysis in accordancewith the present invention.

FIG. 45B plots expected and observed signal intensity versus diffusiontime for the structure shown in FIG. 45A.

FIG. 46A shows a simplified plan view of an alternative embodiment of amicrofluidic structure for performing diffusion analysis in accordancewith the present invention.

FIG. 46B plots expected and observed signal intensity versus diffusiondistance for the structure shown in FIG. 46A.

FIG. 47 shows a plan view of the creation of a microfluidic freeinterface between a flowing fluid and a dead-ended branch channel.

FIG. 48 shows a plan view of diffusion of nutrients and waste across amicrofluidic free interface to and from a cell cage.

FIG. 49 shows a simplified plan view of one embodiment of a microfluidicstructure in accordance with the present invention for performing acompetitive binding assay.

FIG. 50 shows a simplified plan view of an alternative embodiment of amicrofluidic structure in accordance with the present invention forperforming a competitive binding assay.

FIG. 51 shows a simplified plan view of an embodiment of a microfluidicstructure in accordance with the present invention for performing asubstrate turnover assay.

FIG. 52 shows a simplified plan view of one embodiment of a microfluidicstructure in accordance with the present invention for creatingdiffusion gradients of two different species in different dimensions.

FIG. 53 shows a simplified plan view of an alternative embodiment of amicrofluidic structure in accordance with the present invention forcreating diffusion gradients of two different species in differentdimensions.

FIG. 54 plots Log(R/B) vs. number of slugs injected for one embodimentof a cross-flow injection system in accordance with the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

Portions of the prior discussion have referenced the use of microfluidicstructures to accomplish free interface diffusion in accordance withembodiments of the present invention. The following discussion relatesto formation of microfabricated fluidic devices utilizing elastomermaterials, as described generally in U.S. patent applications 09/826,585filed Apr. 6, 2001, Ser. No. 09/724,784 filed Nov. 28, 2000, and Ser.No. 09/605,520, filed Jun. 27, 2000. These patent applications arehereby incorporated by reference.

I. Microfabricated Elastomer Structures

1. Methods of Fabricating

Exemplary methods of fabricating the present invention are providedherein. It is to be understood that the present invention is not limitedto fabrication by one or the other of these methods. Rather, othersuitable methods of fabricating the present microstructures, includingmodifying the present methods, are also contemplated.

FIGS. 1 to 7B illustrate sequential steps of a first preferred method offabricating the present microstructure, (which may be used as a pump orvalve). FIGS. 8 to 18 illustrate sequential steps of a second preferredmethod of fabricating the present microstructure, (which also may beused as a pump or valve).

As will be explained, the preferred method of FIGS. 1 to 7B involvesusing pre-cured elastomer layers which are assembled and bonded. In analternative method, each layer of elastomer may be cured “in place”. Inthe following description “channel” refers to a recess in theelastomeric structure which can contain a flow of fluid or gas.

Referring to FIG. 1, a first micro-machined mold 10 is provided.Micro-machined mold 10 may be fabricated by a number of conventionalsilicon processing methods, including but not limited tophotolithography, ion-milling, and electron beam lithography.

As can be seen, micro-machined mold 10 has a raised line or protrusion11 extending there along. A first elastomeric layer 20 is cast on top ofmold 10 such that a first recess 21 will be formed in the bottom surfaceof elastomeric layer 20, (recess 21 corresponding in dimension toprotrusion 11), as shown.

As can be seen in FIG. 2, a second micro-machined mold 12 having araised protrusion 13 extending there along is also provided. A secondelastomeric layer 22 is cast on top of mold 12, as shown, such that arecess 23 will be formed in its bottom surface corresponding to thedimensions of protrusion 13.

As can be seen in the sequential steps illustrated in FIGS. 3 and 4,second elastomeric layer 22 is then removed from mold 12 and placed ontop of first elastomeric layer 20. As can be seen, recess 23 extendingalong the bottom surface of second elastomeric layer 22 will form a flowchannel 32.

Referring to FIG. 5, the separate first and second elastomeric layers 20and 22 (FIG. 4) are then bonded together to form an integrated (i.e.:monolithic) elastomeric structure 24.

As can been seen in the sequential step of FIGS. 6 and 7A, elastomericstructure 24 is then removed from mold 10 and positioned on top of aplanar substrate 14. As can be seen in FIGS. 7A and 7B, when elastomericstructure 24 has been sealed at its bottom surface to planar substrate14, recess 21 will form a flow channel 30.

The present elastomeric structures form a reversible hermetic seal withnearly any smooth planar substrate. An advantage to forming a seal thisway is that the elastomeric structures may be peeled up, washed, andre-used. In preferred aspects, planar substrate 14 is glass. A furtheradvantage of using glass is that glass is transparent, allowing opticalinterrogation of elastomer channels and reservoirs. Alternatively, theelastomeric structure may be bonded onto a flat elastomer layer by thesame method as described above, forming a permanent and high-strengthbond. This may prove advantageous when higher back pressures are used.

As can be seen in FIGS. 7A and 7B, flow channels 30 and 32 arepreferably disposed at an angle to one another with a small membrane 25of substrate 24 separating the top of flow channel 30 from the bottom offlow channel 32.

In preferred aspects, planar substrate 14 is glass. An advantage ofusing glass is that the present elastomeric structures may be peeled up,washed and reused. A further advantage of using glass is that opticalsensing may be employed. Alternatively, planar substrate 14 may be anelastomer itself, which may prove advantageous when higher backpressures are used.

The method of fabrication just described may be varied to form astructure having a membrane composed of an elastomeric materialdifferent than that forming the walls of the channels of the device.This variant fabrication method is illustrated in FIGS. 7C-G.

Referring to FIG. 7C, a first micro-machined mold 10 is provided.Micro-machined mold 10 has a raised line or protrusion 11 extendingthere along. In FIG. 7D, first elastomeric layer 20 is cast on top offirst micro-machined mold 10 such that the top of the first elastomericlayer 20 is flush with the top of raised line or protrusion 11. This maybe accomplished by carefully controlling the volume of elastomericmaterial spun onto mold 10 relative to the known height of raised line11. Alternatively, the desired shape could be formed by injectionmolding.

In FIG. 7E, second micro-machined mold 12 having a raised protrusion 13extending there along is also provided. Second elastomeric layer 22 iscast on top of second mold 12 as shown, such that recess 23 is formed inits bottom surface corresponding to the dimensions of protrusion 13.

In FIG. 7F, second elastomeric layer 22 is removed from mold 12 andplaced on top of third elastomeric layer 222. Second elastomeric layer22 is bonded to third elastomeric layer 20 to form integral elastomericblock 224 using techniques described in detail below. At this point inthe process, recess 23 formerly occupied by raised line 13 will formflow channel 23.

In FIG. 7G, elastomeric block 224 is placed on top of firstmicro-machined mold 10 and first elastomeric layer 20. Elastomeric blockand first elastomeric layer 20 are then bonded together to form anintegrated (i.e.: monolithic) elastomeric structure 24 having a membranecomposed of a separate elastomeric layer 222.

When elastomeric structure 24 has been sealed at its bottom surface to aplanar substrate in the manner described above in connection with FIG.7A, the recess formerly occupied by raised line 11 will form flowchannel 30.

The variant fabrication method illustrated above in conjunction withFIGS. 7C-G offers the advantage of permitting the membrane portion to becomposed of a separate material than the elastomeric material of theremainder of the structure. This is important because the thickness andelastic properties of the membrane play a key role in operation of thedevice. Moreover, this method allows the separate elastomer layer toreadily be subjected to conditioning prior to incorporation into theelastomer structure. As discussed in detail below, examples ofpotentially desirable condition include the introduction of magnetic orelectrically conducting species to permit actuation of the membrane,and/or the introduction of dopant into the membrane in order to alterits elasticity.

While the above method is illustrated in connection with forming variousshaped elastomeric layers formed by replication molding on top of amicromachined mold, the present invention is not limited to thistechnique. Other techniques could be employed to form the individuallayers of shaped elastomeric material that are to be bonded together.For example, a shaped layer of elastomeric material could be formed bylaser cutting or injection molding, or by methods utilizing chemicaletching and/or sacrificial materials as discussed below in conjunctionwith the second exemplary method.

An alternative method fabricates a patterned elastomer structureutilizing development of photoresist encapsulated within elastomermaterial. However, the methods in accordance with the present inventionare not limited to utilizing photoresist. Other materials such as metalscould also serve as sacrificial materials to be removed selective to thesurrounding elastomer material, and the method would remain within thescope of the present invention. For example, gold metal may be etchedselective to RTV 615 elastomer utilizing the appropriate chemicalmixture.

2. Layer And Channel Dimensions

Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

In preferred aspects, flow channels 30, 32, 60 and 62 preferably havewidth-to-depth ratios of about 10:1. A non-exclusive list of otherranges of width-to-depth ratios in accordance with embodiments of thepresent invention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels 30, 32, 60 and 62 have widths of about 1 to 1000microns. A non-exclusive list of other ranges of widths of flow channelsin accordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

Flow channels 30, 32, 60, and 62 have depths of about 1 to 100 microns.A non-exclusive list of other ranges of depths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250microns, and more preferably 1 to 100 microns, more preferably 2 to 20microns, and most preferably 5 to 10 microns. Exemplary channel depthsinclude including 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1μm, 2 μm, 3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm,and 250 μm.

The flow channels are not limited to these specific dimension ranges andexamples given above, and may vary in width in order to affect themagnitude of force required to deflect the membrane as discussed atlength below in conjunction with FIG. 27. For example, extremely narrowflow channels having a width on the order of 0.01 μm may be useful inoptical and other applications, as discussed in detail below.Elastomeric structures which include portions having channels of evengreater width than described above are also contemplated by the presentinvention, and examples of applications of utilizing such wider flowchannels include fluid reservoir and mixing channel structures.

The Elastomeric layers may be cast thick for mechanical stability. In anexemplary embodiment, elastomeric layer 22 of FIG. 1 is 50 microns toseveral centimeters thick, and more preferably approximately 4 mm thick.A non-exclusive list of ranges of thickness of the elastomer layer inaccordance with other embodiments of the present invention is betweenabout 0.1 micron to 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100microns to 10 mm.

Accordingly, membrane 25 of FIG. 7B separating flow channels 30 and 32has a typical thickness of between about 0.01 and 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250, morepreferably 1 to 100 microns, more preferably 2 to 50 microns, and mostpreferably 5 to 40 microns. As such, the thickness of elastomeric layer22 is about 100 times the thickness of elastomeric layer 20. Exemplarymembrane thicknesses include 0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm,0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm,15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 750 μm, and 1000 μm.

3. Soft Lithographic Bonding

Preferably, elastomeric layers are bonded together chemically, usingchemistry that is intrinsic to the polymers comprising the patternedelastomer layers. Most preferably, the bonding comprises two component“addition cure” bonding.

In a preferred aspect, the various layers of elastomer are boundtogether in a heterogeneous bonding in which the layers have a differentchemistry. Alternatively, a homogenous bonding may be used in which alllayers would be of the same chemistry. Thirdly, the respective elastomerlayers may optionally be glued together by an adhesive instead. In afourth aspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

Alternatively in a heterogeneous aspect, the elastomeric layers arecomposed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogeneous aspect, the bonding process usedto bind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

In an exemplary aspect of the present invention, elastomeric structuresare formed utilizing Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical.

In one embodiment in accordance with the present invention, two-layerelastomeric structures were fabricated from pure acrylated Urethane Ebe270. A thin bottom layer was spin coated at 8000 rpm for 15 seconds at170° C. The top and bottom layers were initially cured under ultravioletlight for 10 minutes under nitrogen utilizing a Model ELC 500 devicemanufactured by Electrolite corporation. The assembled layers were thencured for an additional 30 minutes. Reaction was catalyzed by a 0.5%vol/vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals.The resulting elastomeric material exhibited moderate elasticity andadhesion to glass.

In another embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from a combination of25% Ebe 270/50% Irr245/25% isopropyl alcohol for a thin bottom layer,and pure acrylated Urethane Ebe 270 as a top layer. The thin bottomlayer was initially cured for 5 min, and the top layer initially curedfor 10 minutes, under ultraviolet light under nitrogen utilizing a ModelELC 500 device manufactured by Electrolite corporation. The assembledlayers were then cured for an additional 30 minutes. Reaction wascatalyzed by a 0.5% vol/vol mixture of Irgacure 500 manufactured byCiba-Geigy Chemicals. The resulting elastomeric material exhibitedmoderate elasticity and adhered to glass.

Alternatively, other bonding methods may be used, including activatingthe elastomer surface, for example by plasma exposure, so that theelastomer layers/substrate will bond when placed in contact. Forexample, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly (dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974-4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Where encapsulation of sacrificial layers is employed to fabricate theelastomer structure, bonding of successive elastomeric layers may beaccomplished by pouring uncured elastomer over a previously curedelastomeric layer and any sacrificial material patterned thereupon.Bonding between elastomer layers occurs due to interpenetration andreaction of the polymer chains of an uncured elastomer layer with thepolymer chains of a cured elastomer layer. Subsequent curing of theelastomeric layer will create a bond between the elastomeric layers andcreate a monolithic elastomeric structure.

Referring to the first method of FIGS. 1 to 7B, first elastomeric layer20 may be created by spin-coating an RTV mixture on microfabricated mold12 at 2000 rpm's for 30 seconds yielding a thickness of approximately 40microns. Second elastomeric layer 22 may be created by spin-coating anRTV mixture on microfabricated mold 11. Both layers 20 and 22 may beseparately baked or cured at about 80° C. for 1.5 hours. The secondelastomeric layer 22 may be bonded onto first elastomeric layer 20 atabout 80° C. for about 1.5 hours.

Micromachined molds 10 and 12 may be patterned photoresist on siliconwafers. In an exemplary aspect, a Shipley SJR 5740 photoresist was spunat 2000 rpm patterned with a high resolution transparency film as a maskand then developed yielding an inverse channel of approximately 10microns in height. When baked at approximately 200° C. for about 30minutes, the photoresist reflows and the inverse channels becomerounded. In preferred aspects, the molds may be treated withtrimethylchlorosilane (TMCS) vapor for about a minute before each use inorder to prevent adhesion of silicone rubber.

4. Suitable Elastomeric Materials

Allcock et al, Contemporary Polymer Chemistry, 2^(nd) Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.Elastomeric materials having a Young's modulus of between about 1 Pa-1TPa, more preferably between about 10 Pa-100 GPa, more preferablybetween about 20 Pa-1 GPa, more preferably between about 50 Pa-10 MPa,and more preferably between about 100 Pa-1 MPa are useful in accordancewith the present invention, although elastomeric materials having aYoung's modulus outside of these ranges could also be utilized dependingupon the needs of a particular application.

The systems of the present invention may be fabricated from a widevariety of elastomers. In an exemplary aspect, the elastomeric layersmay preferably be fabricated from silicone rubber. However, othersuitable elastomers may also be used.

In an exemplary aspect of the present invention, the present systems arefabricated from an elastomeric polymer such as GE RTV 615 (formulation),a vinyl-silane crosslinked (type) silicone elastomer (family). However,the present systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves, or they may be of twodifferent types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability.

There are many, many types of elastomeric polymers. A brief descriptionof the most common classes of elastomers is presented here, with theintent of showing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones.

Polyisoprene, polybutadiene, polychloroprene:

Polyisoprene, polybutadiene, and polychloroprene are all polymerizedfrom diene monomers, and therefore have one double bond per monomer whenpolymerized. This double bond allows the polymers to be converted toelastomers by vulcanization (essentially, sulfur is used to formcrosslinks between the double bonds by heating). This would easily allowhomogeneous multilayer soft lithography by incomplete vulcanization ofthe layers to be bonded; photoresist encapsulation would be possible bya similar mechanism.

Polyisobutylene:

Pure Polyisobutylene has no double bonds, but is crosslinked to use asan elastomer by including a small amount (˜1%) of isoprene in thepolymerization. The isoprene monomers give pendant double bonds on thePolyisobutylene backbone, which may then be vulcanized as above.

Poly(styrene-butadiene-styrene):

Poly(styrene-butadiene-styrene) is produced by living anionicpolymerization (that is, there is no natural chain-terminating step inthe reaction), so “live” polymer ends can exist in the cured polymer.This makes it a natural candidate for the present photoresistencapsulation system (where there will be plenty of unreacted monomer inthe liquid layer poured on top of the cured layer). Incomplete curingwould allow homogeneous multilayer soft lithography (A to A bonding).The chemistry also facilitates making one layer with extra butadiene(“A”) and coupling agent and the other layer (“B”) with a butadienedeficit (for heterogeneous multilayer soft lithography). SBS is a“thermoset elastomer”, meaning that above a certain temperature it meltsand becomes plastic (as opposed to elastic); reducing the temperatureyields the elastomer again. Thus, layers can be bonded together byheating.

Polyurethanes:

Polyurethanes are produced from di-isocyanates (A-A) and di-alcohols ordi-amines (B-B); since there are a large variety of di-isocyanates anddi-alcohols/amines, the number of different types of polyurethanes ishuge. The A vs. B nature of the polymers, however, would make themuseful for heterogeneous multilayer soft lithography just as RTV 615 is:by using excess A-A in one layer and excess B-B in the other layer.

Silicones:

Silicone polymers probably have the greatest structural variety, andalmost certainly have the greatest number of commercially availableformulations. The vinyl-to-(Si—H) crosslinking of RTV 615 (which allowsboth heterogeneous multilayer soft lithography and photoresistencapsulation) has already been discussed, but this is only one ofseveral crosslinking methods used in silicone polymer chemistry.

5. Operation of Device

FIGS. 7B and 7H together show the closing of a first flow channel bypressurizing a second flow channel, with FIG. 7B (a front sectional viewcutting through flow channel 32 in corresponding FIG. 7A), showing anopen first flow channel 30; with FIG. 7H showing first flow channel 30closed by pressurization of the second flow channel 32.

Referring to FIG. 7B, first flow channel 30 and second flow channel 32are shown. Membrane 25 separates the flow channels, forming the top offirst flow channel 30 and the bottom of second flow channel 32. As canbe seen, flow channel 30 is “open”.

As can be seen in FIG. 7H, pressurization of flow channel 32 (either bygas or liquid introduced therein) causes membrane 25 to deflectdownward, thereby pinching off flow F passing through flow channel 30.Accordingly, by varying the pressure in channel 32, a linearly actuablevalving system is provided such that flow channel 30 can be opened orclosed by moving membrane 25 as desired. (For illustration purposesonly, channel 30 in FIG. 7G is shown in a “mostly closed” position,rather than a “fully closed” position).

Since such valves are actuated by moving the roof of the channelsthemselves (i.e.: moving membrane 25) valves and pumps produced by thistechnique have a truly zero dead volume, and switching valves made bythis technique have a dead volume approximately equal to the activevolume of the valve, for example about 100×100×10 μm=100 pL. Such deadvolumes and areas consumed by the moving membrane are approximately twoorders of magnitude smaller than known conventional microvalves. Smallerand larger valves and switching valves are contemplated in the presentinvention, and a non-exclusive list of ranges of dead volume includes 1aL to 1 uL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL, and 1 pL to100 pL.

The extremely small volumes capable of being delivered by pumps andvalves in accordance with the present invention represent a substantialadvantage. Specifically, the smallest known volumes of fluid capable ofbeing manually metered is around 0.1 μl. The smallest known volumescapable of being metered by automated systems is about ten-times larger(1 μl). Utilizing pumps and valves in accordance with the presentinvention, volumes of liquid of 10 nl or smaller can routinely bemetered and dispensed. The accurate metering of extremely small volumesof fluid enabled by the present invention would be extremely valuable ina large number of biological applications, including diagnostic testsand assays.

Equation 1 represents a highly simplified mathematical model ofdeflection of a rectangular, linear, elastic, isotropic plate of uniformthickness by an applied pressure:

w=(BPb ⁴)/(Eh ³),  (1)

where:

-   -   w=deflection of plate;    -   B=shape coefficient (dependent upon length vs. width and support        of edges of plate);    -   P=applied pressure;    -   b=plate width    -   E=Young's modulus; and    -   h=plate thickness.

Thus even in this extremely simplified expression, deflection of anelastomeric membrane in response to a pressure will be a function of:the length, width, and thickness of the membrane, the flexibility of themembrane (Young's modulus), and the applied actuation force. Becauseeach of these parameters will vary widely depending upon the actualdimensions and physical composition of a particular elastomeric devicein accordance with the present invention, a wide range of membranethicknesses and elasticity's, channel widths, and actuation forces arecontemplated by the present invention.

It should be understood that the formula just presented is only anapproximation, since in general the membrane does not have uniformthickness, the membrane thickness is not necessarily small compared tothe length and width, and the deflection is not necessarily smallcompared to length, width, or thickness of the membrane. Nevertheless,the equation serves as a useful guide for adjusting variable parametersto achieve a desired response of deflection versus applied force.

FIGS. 8A and 8B illustrate valve opening vs. applied pressure for a 100μm wide first flow channel 30 and a 50 μm wide second flow channel 32.The membrane of this device was formed by a layer of General ElectricSilicones RTV 615 having a thickness of approximately 30 μm and aYoung's modulus of approximately 750 kPa. FIGS. 21A and 21B show theextent of opening of the valve to be substantially linear over most ofthe range of applied pressures.

Air pressure was applied to actuate the membrane of the device through a10 cm long piece of plastic tubing having an outer diameter of 0.025″connected to a 25 mm piece of stainless steel hypodermic tubing with anouter diameter of 0.025″ and an inner diameter of 0.013″. This tubingwas placed into contact with the control channel by insertion into theelastomeric block in a direction normal to the control channel. Airpressure was applied to the hypodermic tubing from an external LHDAminiature solenoid valve manufactured by Lee Co.

While control of the flow of material through the device has so far beendescribed utilizing applied gas pressure, other fluids could be used.

For example, air is compressible, and thus experiences some finite delaybetween the time of application of pressure by the external solenoidvalve and the time that this pressure is experienced by the membrane. Inan alternative embodiment of the present invention, pressure could beapplied from an external source to a noncompressible fluid such as wateror hydraulic oils, resulting in a near-instantaneous transfer of appliedpressure to the membrane. However, if the displaced volume of the valveis large or the control channel is narrow, higher viscosity of a controlfluid may contribute to delay in actuation. The optimal medium fortransferring pressure will therefore depend upon the particularapplication and device configuration, and both gaseous and liquid mediaare contemplated by the invention.

While external applied pressure as described above has been applied by apump/tank system through a pressure regulator and external miniaturevalve, other methods of applying external pressure are also contemplatedin the present invention, including gas tanks, compressors, pistonsystems, and columns of liquid. Also contemplated is the use ofnaturally occurring pressure sources such as may be found inside livingorganisms, such as blood pressure, gastric pressure, the pressurepresent in the cerebro-spinal fluid, pressure present in theintra-ocular space, and the pressure exerted by muscles during normalflexure. Other methods of regulating external pressure are alsocontemplated, such as miniature valves, pumps, macroscopic peristalticpumps, pinch valves, and other types of fluid regulating equipment suchas is known in the art.

As can be seen, the response of valves in accordance with embodiments ofthe present invention have been experimentally shown to be almostperfectly linear over a large portion of its range of travel, withminimal hysteresis. Accordingly, the present valves are ideally suitedfor microfluidic metering and fluid control. The linearity of the valveresponse demonstrates that the individual valves are well modeled asHooke's Law springs. Furthermore, high pressures in the flow channel(i.e.: back pressure) can be countered simply by increasing theactuation pressure. Experimentally, the present inventors have achievedvalve closure at back pressures of 70 kPa, but higher pressures are alsocontemplated. The following is a nonexclusive list of pressure rangesencompassed by the present invention: 10 Pa-25 MPa; 100 Pa-10 Mpa, 1kPa-1 MPa, 1 kPa-300 kPa, 5 kPa-200 kPa, and 15 kPa-100 kPa.

While valves and pumps do not require linear actuation to open andclose, linear response does allow valves to more easily be used asmetering devices. In one embodiment of the invention, the opening of thevalve is used to control flow rate by being partially actuated to aknown degree of closure. Linear valve actuation makes it easier todetermine the amount of actuation force required to close the valve to adesired degree of closure. Another benefit of linear actuation is thatthe force required for valve actuation may be easily determined from thepressure in the flow channel. If actuation is linear, increased pressurein the flow channel may be countered by adding the same pressure (forceper unit area) to the actuated portion of the valve.

Linearity of a valve depends on the structure, composition, and methodof actuation of the valve structure. Furthermore, whether linearity is adesirable characteristic in a valve depends on the application.Therefore, both linearly and non-linearly actuable valves arecontemplated in the present invention, and the pressure ranges overwhich a valve is linearly actuable will vary with the specificembodiment.

FIG. 9 illustrates time response (i.e.: closure of valve as a functionof time in response to a change in applied pressure) of a 100 μm×100μm×10 μm RTV microvalve with 10-cm-long air tubing connected from thechip to a pneumatic valve as described above.

Two periods of digital control signal, actual air pressure at the end ofthe tubing and valve opening are shown in FIG. 9. The pressure appliedon the control line is 100 kPa, which is substantially higher than the˜40 kPa required to close the valve. Thus, when closing, the valve ispushed closed with a pressure 60 kPa greater than required. Whenopening, however, the valve is driven back to its rest position only byits own spring force 40 kPa). Thus, τ_(close) is expected to be smallerthan τ_(open). There is also a lag between the control signal andcontrol pressure response, due to the limitations of the miniature valveused to control the pressure. Calling such lags t and the 1/e timeconstants τ, the values are: t_(open)=3.63 ms, τ_(open)=1.88 ms,t_(close)=2.15 ms, τ_(close)=0.51 ms. If 3τ each are allowed for openingand closing, the valve runs comfortably at 75 Hz when filled withaqueous solution.

If one used another actuation method which did not suffer from openingand closing lag, this valve would run at ˜375 Hz. Note also that thespring constant can be adjusted by changing the membrane thickness; thisallows optimization for either fast opening or fast closing. The springconstant could also be adjusted by changing the elasticity (Young'smodulus) of the membrane, as is possible by introducing dopant into themembrane or by utilizing a different elastomeric material to serve asthe membrane (described above in conjunction with FIGS. 7C-H).

When experimentally measuring the valve properties as illustrated inFIG. 9 the valve opening was measured by fluorescence. In theseexperiments, the flow channel was filled with a solution of fluoresceinisothiocyanate (FITC) in buffer (pH 8) and the fluorescence of a squarearea occupying the center ˜⅓rd of the channel is monitored on anepi-fluorescence microscope with a photomultiplier tube with a 10 kHzbandwidth. The pressure was monitored with a Wheatstone-bridge pressuresensor (SenSym SCC15GD2) pressurized simultaneously with the controlline through nearly identical pneumatic connections.

6. Flow Channel Cross Sections

The flow channels of the present invention may optionally be designedwith different cross sectional sizes and shapes, offering differentadvantages, depending upon their desired application. For example, thecross sectional shape of the lower flow channel may have a curved uppersurface, either along its entire length or in the region disposed underan upper cross channel). Such a curved upper surface facilitates valvesealing, as follows.

Referring to FIG. 10, a cross sectional view (similar to that of FIG.7B) through flow channels 30 and 32 is shown. As can be seen, flowchannel 30 is rectangular in cross sectional shape. In an alternatepreferred aspect of the invention, as shown in FIG. 10, thecross-section of a flow channel 30 instead has an upper curved surface.

Referring first to FIG. 10, when flow channel 32 is pressurized, themembrane portion 25 of elastomeric block 24 separating flow channels 30and 32 will move downwardly to the successive positions shown by thedotted lines 25A, 25B, 25C, 25D, and 25E. As can be seen, incompletesealing may possibly result at the edges of flow channel 30 adjacentplanar substrate 14.

In the alternate preferred embodiment of FIG. 11, flow channel 30 a hasa curved upper wall 25A. When flow channel 32 is pressurized, membraneportion 25 will move downwardly to the successive positions shown bydotted lines 25A2, 25A3, 25A4 and 25A5, with edge portions of themembrane moving first into the flow channel, followed by top membraneportions. An advantage of having such a curved upper surface at membrane25A is that a more complete seal will be provided when flow channel 32is pressurized. Specifically, the upper wall of the flow channel 30 willprovide a continuous contacting edge against planar substrate 14,thereby avoiding the “island” of contact seen between wall 25 and thebottom of flow channel 30 in FIG. 10.

Another advantage of having a curved upper flow channel surface atmembrane 25A is that the membrane can more readily conform to the shapeand volume of the flow channel in response to actuation. Specifically,where a rectangular flow channel is employed, the entire perimeter (2×flow channel height, plus the flow channel width) must be forced intothe flow channel. However where an arched flow channel is used, asmaller perimeter of material (only the semi-circular arched portion)must be forced into the channel. In this manner, the membrane requiresless change in perimeter for actuation and is therefore more responsiveto an applied actuation force to block the flow channel.

In an alternate aspect, (not illustrated), the bottom of flow channel 30is rounded such that its curved surface mates with the curved upper wall25A as seen in FIG. 20 described above.

In summary, the actual conformational change experienced by the membraneupon actuation will depend upon the configuration of the particularelastomeric structure. Specifically, the conformational change willdepend upon the length, width, and thickness profile of the membrane,its attachment to the remainder of the structure, and the height, width,and shape of the flow and control channels and the material propertiesof the elastomer used. The conformational change may also depend uponthe method of actuation, as actuation of the membrane in response to anapplied pressure will vary somewhat from actuation in response to amagnetic or electrostatic force.

Moreover, the desired conformational change in the membrane will alsovary depending upon the particular application for the elastomericstructure. In the simplest embodiments described above, the valve mayeither be open or closed, with metering to control the degree of closureof the valve. In other embodiments however, it may be desirable to alterthe shape of the membrane and/or the flow channel in order to achievemore complex flow regulation. For instance, the flow channel could beprovided with raised protrusions beneath the membrane portion, such thatupon actuation the membrane shuts off only a percentage of the flowthrough the flow channel, with the percentage of flow blockedinsensitive to the applied actuation force.

Many membrane thickness profiles and flow channel cross-sections arecontemplated by the present invention, including rectangular,trapezoidal, circular, ellipsoidal, parabolic, hyperbolic, andpolygonal, as well as sections of the above shapes. More complexcross-sectional shapes, such as the embodiment with protrusionsdiscussed immediately above or an embodiment having concavities in theflow channel, are also contemplated by the present invention.

In addition, while the invention is described primarily above inconjunction with an embodiment wherein the walls and ceiling of the flowchannel are formed from elastomer, and the floor of the channel isformed from an underlying substrate, the present invention is notlimited to this particular orientation. Walls and floors of channelscould also be formed in the underlying substrate, with only the ceilingof the flow channel constructed from elastomer. This elastomer flowchannel ceiling would project downward into the channel in response toan applied actuation force, thereby controlling the flow of materialthrough the flow channel. In general, monolithic elastomer structures asdescribed elsewhere in the instant application are preferred formicrofluidic applications. However, it may be useful to employ channelsformed in the substrate where such an arrangement provides advantages.For instance, a substrate including optical waveguides could beconstructed so that the optical waveguides direct light specifically tothe side of a microfluidic channel.

7. Alternate Valve Actuation Techniques

In addition to pressure based actuation systems described above,optional electrostatic and magnetic actuation systems are alsocontemplated, as follows.

Electrostatic actuation can be accomplished by forming oppositelycharged electrodes (which will tend to attract one another when avoltage differential is applied to them) directly into the monolithicelastomeric structure. For example, referring to FIG. 7B, an optionalfirst electrode 70 (shown in phantom) can be positioned on (or in)membrane 25 and an optional second electrode 72 (also shown in phantom)can be positioned on (or in) planar substrate 14. When electrodes 70 and72 are charged with opposite polarities, an attractive force between thetwo electrodes will cause membrane 25 to deflect downwardly, therebyclosing the “valve” (i.e.: closing flow channel 30).

For the membrane electrode to be sufficiently conductive to supportelectrostatic actuation, but not so mechanically stiff so as to impedethe valve's motion, a sufficiently flexible electrode must be providedin or over membrane 25. Such an electrode may be provided by a thinmetallization layer, doping the polymer with conductive material, ormaking the surface layer out of a conductive material.

In an exemplary aspect, the electrode present at the deflecting membranecan be provided by a thin metallization layer which can be provided, forexample, by sputtering a thin layer of metal such as 20 nm of gold. Inaddition to the formation of a metallized membrane by sputtering, othermetallization approaches such as chemical epitaxy, evaporation,electroplating, and electroless plating are also available. Physicaltransfer of a metal layer to the surface of the elastomer is alsoavailable, for example by evaporating a metal onto a flat substrate towhich it adheres poorly, and then placing the elastomer onto the metaland peeling the metal off of the substrate.

A conductive electrode 70 may also be formed by depositing carbon black(i.e. Cabot Vulcan XC72R) on the elastomer surface, either by wiping onthe dry powder or by exposing the elastomer to a suspension of carbonblack in a solvent which causes swelling of the elastomer, (such as achlorinated solvent in the case of PDMS). Alternatively, the electrode70 may be formed by constructing the entire layer 20 out of elastomerdoped with conductive material (i.e. carbon black or finely dividedmetal particles). Yet further alternatively, the electrode may be formedby electrostatic deposition, or by a chemical reaction that producescarbon. In experiments conducted by the present inventors, conductivitywas shown to increase with carbon black concentration from 5.6×10⁻¹⁶ toabout 5×10⁻³ (Ω-cm)⁻¹. The lower electrode 72, which is not required tomove, may be either a compliant electrode as described above, or aconventional electrode such as evaporated gold, a metal plate, or adoped semiconductor electrode.

Magnetic actuation of the flow channels can be achieved by fabricatingthe membrane separating the flow channels with a magneticallypolarizable material such as iron, or a permanently magnetized materialsuch as polarized NdFeB. In experiments conducted by the presentinventors, magnetic silicone was created by the addition of iron powder(about 1 um particle size), up to 20% iron by weight.

Where the membrane is fabricated with a magnetically polarizablematerial, the membrane can be actuated by attraction in response to anapplied magnetic field Where the membrane is fabricated with a materialcapable of maintaining permanent magnetization, the material can firstbe magnetized by exposure to a sufficiently high magnetic field, andthen actuated either by attraction or repulsion in response to thepolarity of an applied inhomogeneous magnetic field.

The magnetic field causing actuation of the membrane can be generated ina variety of ways. In one embodiment, the magnetic field is generated byan extremely small inductive coil formed in or proximate to theelastomer membrane. The actuation effect of such a magnetic coil wouldbe localized, allowing actuation of individual pump and/or valvestructures. Alternatively, the magnetic field could be generated by alarger, more powerful source, in which case actuation would be globaland would actuate multiple pump and/or valve structures at one time.

It is also possible to actuate the device by causing a fluid flow in thecontrol channel based upon the application of thermal energy, either bythermal expansion or by production of gas from liquid. For example, inone alternative embodiment in accordance with the present invention, apocket of fluid (e.g. in a fluid-filled control channel) is positionedover the flow channel. Fluid in the pocket can be in communication witha temperature variation system, for example a heater. Thermal expansionof the fluid, or conversion of material from the liquid to the gasphase, could result in an increase in pressure, closing the adjacentflow channel. Subsequent cooling of the fluid would relieve pressure andpermit the flow channel to open.

8. Networked Systems

FIGS. 12A and 12B show a views of a single on/off valve, identical tothe systems set forth above, (for example in FIG. 7A). FIGS. 13A and 13Bshows a peristaltic pumping system comprised of a plurality of thesingle addressable on/off valves as seen in FIG. 12, but networkedtogether. FIG. 14 is a graph showing experimentally achieved pumpingrates vs. frequency for the peristaltic pumping system of FIG. 13. FIGS.15A and 15B show a schematic view of a plurality of flow channels whichare controllable by a single control line. This system is also comprisedof a plurality of the single addressable on/off valves of FIG. 12,multiplexed together, but in a different arrangement than that of FIG.12. FIG. 16 is a schematic illustration of a multiplexing system adaptedto permit fluid flow through selected channels, comprised of a pluralityof the single on/off valves of FIG. 12, joined or networked together.

Referring first to FIGS. 12A and 12B, a schematic of flow channels 30and 32 is shown. Flow channel 30 preferably has a fluid (or gas) flow Fpassing therethrough. Flow channel 32, (which crosses over flow channel30, as was already explained herein), is pressurized such that membrane25 separating the flow channels may be depressed into the path of flowchannel 30, shutting off the passage of flow F therethrough, as has beenexplained. As such, “flow channel” 32 can also be referred to as a“control line” which actuates a single valve in flow channel 30. InFIGS. 12 to 15, a plurality of such addressable valves are joined ornetworked together in various arrangements to produce pumps, capable ofperistaltic pumping, and other fluidic logic applications.

Referring to FIGS. 13A and 13B, a system for peristaltic pumping isprovided, as follows. A flow channel 30 has a plurality of generallyparallel flow channels (i.e.: control lines) 32A, 32B and 32C passingthereover. By pressurizing control line 32A, flow F through flow channel30 is shut off under membrane 25A at the intersection of control line32A and flow channel 30. Similarly, (but not shown), by pressurizingcontrol line 32B, flow F through flow channel 30 is shut off undermembrane 25B at the intersection of control line 32B and flow channel30, etc.

Each of control lines 32A, 32B, and 32C is separately addressable.Therefore, peristalsis may be actuated by the pattern of actuating 32Aand 32C together, followed by 32A, followed by 32A and 32B together,followed by 32B, followed by 32B and C together, etc. This correspondsto a successive “101, 100, 110, 010, 011, 001” pattern, where “0”indicates “valve open” and “1” indicates “valve closed.” Thisperistaltic pattern is also known as a 120° pattern (referring to thephase angle of actuation between three valves). Other peristalticpatterns are equally possible, including 60° and 90° patterns.

In experiments performed by the inventors, a pumping rate of 2.35 nL/swas measured by measuring the distance traveled by a column of water inthin (0.5 mm i.d.) tubing; with 100×100×10 μm valves under an actuationpressure of 40 kPa. The pumping rate increased with actuation frequencyuntil approximately 75 Hz, and then was nearly constant until above 200Hz. The valves and pumps are also quite durable and the elastomermembrane, control channels, or bond have never been observed to fail. Inexperiments performed by the inventors, none of the valves in theperistaltic pump described herein show any sign of wear or fatigue aftermore than 4 million actuations. In addition to their durability, theyare also gentle. A solution of E. Coli pumped through a channel andtested for viability showed a 94% survival rate.

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for the peristaltic pumping system of FIG. 13.

FIGS. 15A and 15B illustrates another way of assembling a plurality ofthe addressable valves of FIG. 12. Specifically, a plurality of parallelflow channels 30A, 30B, and 30C are provided. Flow channel (i.e.:control line) 32 passes thereover across flow channels 30A, 30B, and30C. Pressurization of control line 32 simultaneously shuts off flowsF1, F2 and F3 by depressing membranes 25A, 25B, and 25C located at theintersections of control line 32 and flow channels 30A, 30B, and 30C.

FIG. 16 is a schematic illustration of a multiplexing system adapted toselectively permit fluid to flow through selected channels, as follows.The downward deflection of membranes separating the respective flowchannels from a control line passing there above (for example, membranes25A, 25B, and 25C in FIGS. 15A and 15B) depends strongly upon themembrane dimensions. Accordingly, by varying the widths of flow channelcontrol line 32 in FIGS. 15A and 15B, it is possible to have a controlline pass over multiple flow channels, yet only actuate (i.e.: seal)desired flow channels. FIG. 16 illustrates a schematic of such a system,as follows.

A plurality of parallel flow channels 30A, 30B, 30C, 30D, 30E and 30Fare positioned under a plurality of parallel control lines 32A, 32B,32C, 32D, 32E and 32F. Control channels 32A, 32B, 32C, 32D, 32E and 32Fare adapted to shut off fluid flows F1, F2, F3, F4, F5 and F6 passingthrough parallel flow channels 30A, 30B, 30C, 30D, 30E and 30F using anyof the valving systems described above, with the following modification.

Each of control lines 32A, 32B, 32C, 32D, 32E and 32F have both wide andnarrow portions. For example, control line 32A is wide in locationsdisposed over flow channels 30A, 30C and 30E. Similarly, control line32B is wide in locations disposed over flow channels 30B, 30D and 30F,and control line 32C is wide in locations disposed over flow channels30A, 30B, 30E and 30F.

At the locations where the respective control line is wide, itspressurization will cause the membrane (25) separating the flow channeland the control line to depress significantly into the flow channel,thereby blocking the flow passage therethrough. Conversely, in thelocations where the respective control line is narrow, membrane (25)will also be narrow. Accordingly, the same degree of pressurization willnot result in membrane (25) becoming depressed into the flow channel(30). Therefore, fluid passage there under will not be blocked.

For example, when control line 32A is pressurized, it will block flowsF1, F3 and F5 in flow channels 30A, 30C and 30E. Similarly, when controlline 32C is pressurized, it will block flows F1, F2, F5 and F6 in flowchannels 30A, 30B, 30E and 30F. As can be appreciated, more than onecontrol line can be actuated at the same time. For example, controllines 32A and 32C can be pressurized simultaneously to block all fluidflow except F4 (with 32A blocking F1, F3 and F5; and 32C blocking F1,F2, F5 and F6).

By selectively pressurizing different control lines (32) both togetherand in various sequences, a great degree of fluid flow control can beachieved. Moreover, by extending the present system to more than sixparallel flow channels (30) and more than four parallel control lines(32), and by varying the positioning of the wide and narrow regions ofthe control lines, very complex fluid flow control systems may befabricated. A property of such systems is that it is possible to turn onany one flow channel out of n flow channels with only 2(log₂n) controllines.

9. Selectively Addressable Reaction Chambers Along Flow Lines

In a further embodiment of the invention, illustrated in FIGS. 17A-D, asystem for selectively directing fluid flow into one more of a pluralityof reaction chambers disposed along a flow line is provided.

FIG. 17A shows a top view of a flow channel 30 having a plurality ofreaction chambers 80A and 80B disposed there along. Preferably flowchannel 30 and reaction chambers 80A and 80B are formed together asrecesses into the bottom surface of a first layer 100 of elastomer.

FIG. 17B shows a bottom plan view of another elastomeric layer 110 withtwo control lines 32A and 32B each being generally narrow, but havingwide extending portions 33A and 33B formed as recesses therein.

As seen in the exploded view of FIG. 17C, and assembled view of FIG.17D, elastomeric layer 110 is placed over elastomeric layer 100. Layers100 and 110 are then bonded together, and the integrated system operatesto selectively direct fluid flow F (through flow channel 30) into eitheror both of reaction chambers 80A and 80B, as follows. Pressurization ofcontrol line 32A will cause the membrane 25 (i.e.: the thin portion ofelastomer layer 100 located below extending portion 33A and over regions82A of reaction chamber 80A) to become depressed, thereby shutting offfluid flow passage in regions 82A, effectively sealing reaction chamber80 from flow channel 30. As can also be seen, extending portion 33A iswider than the remainder of control line 32A. As such, pressurization ofcontrol line 32A will not result in control line 32A sealing flowchannel 30.

As can be appreciated, either or both of control lines 32A and 32B canbe actuated at once. When both control lines 32A and 32B are pressurizedtogether, sample flow in flow channel 30 will enter neither of reactionchambers 80A or 80B.

The concept of selectably controlling fluid introduction into variousaddressable reaction chambers disposed along a flow line (FIGS. 17A-D)can be combined with concept of selectably controlling fluid flowthrough one or more of a plurality of parallel flow lines (FIG. 16) toyield a system in which a fluid sample or samples can be can be sent toany particular reaction chamber in an array of reaction chambers. Anexample of such a system is provided in FIG. 18, in which parallelcontrol channels 32A, 32B and 32C with extending portions 34 (all shownin phantom) selectively direct fluid flows F1 and F2 into any of thearray of reaction wells 80A, 80B, 80C or 80D as explained above; whilepressurization of control lines 32C and 32D selectively shuts off flowsF2 and F1, respectively.

In yet another novel embodiment, fluid passage between parallel flowchannels is possible. Referring to FIG. 19, either or both of controllines 32A or 32D can be depressurized such that fluid flow throughlateral passageways 35 (between parallel flow channels 30A and 30B) ispermitted. In this aspect of the invention, pressurization of controllines 32C and 32D would shut flow channel 30A between 35A and 35B, andwould also shut lateral passageways 35B. As such, flow entering as flowF1 would sequentially travel through 30A, 35A and leave 30B as flow F4.

10. Switchable Flow Arrays

In yet another novel embodiment, fluid passage can be selectivelydirected to flow in either of two perpendicular directions. An exampleof such a “switchable flow array” system is provided in FIGS. 20A-D.FIG. 20A shows a bottom view of a first layer of elastomer 90, (or anyother suitable substrate), having a bottom surface with a pattern ofrecesses forming a flow channel grid defined by an array of solid posts92, each having flow channels passing there around.

In preferred aspects, an additional layer of elastomer is bound to thetop surface of layer 90 such that fluid flow can be selectively directedto move either in direction F1, or perpendicular direction F2. FIG. 20is a bottom view of the bottom surface of the second layer of elastomer95 showing recesses formed in the shape of alternating “vertical”control lines 96 and “horizontal” control lines 94. “Vertical” controllines 96 have the same width there along, whereas “horizontal” controllines 94 have alternating wide and narrow portions, as shown.

Elastomeric layer 95 is positioned over top of elastomeric layer 90 suchthat “vertical” control lines 96 are positioned over posts 92 as shownin FIG. 20C and “horizontal” control lines 94 are positioned with theirwide portions between posts 92, as shown in FIG. 20D.

As can be seen in FIG. 20C, when “vertical” control lines 96 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 98 will be deflected downwardly over the array of flow channelssuch that flow in only able to pass in flow direction F2 (i.e.:vertically), as shown.

As can be seen in FIG. 20D, when “horizontal” control lines 94 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 99 will be deflected downwardly over the array of flow channels,(but only in the regions where they are widest), such that flow in onlyable to pass in flow direction F1 (i.e.: horizontally), as shown.

The design illustrated in FIG. 20 allows a switchable flow array to beconstructed from only two elastomeric layers, with no vertical viaspassing between control lines in different elastomeric layers required.If all vertical flow control lines 94 are connected, they may bepressurized from one input. The same is true for all horizontal flowcontrol lines 96.

11. Normally-Closed Valve Structure

FIGS. 7B and 7H above depict a valve structure in which the elastomericmembrane is moveable from a first relaxed position to a second actuatedposition in which the flow channel is blocked. However, the presentinvention is not limited to this particular valve configuration.

FIGS. 21A-J show a variety of views of a normally-closed valve structurein which the elastomeric membrane is moveable from a first relaxedposition blocking a flow channel, to a second actuated position in whichthe flow channel is open, utilizing a negative control pressure.

FIG. 21A shows a plan view, and FIG. 21B shows a cross sectional viewalong line 42B-42B′, of normally-closed valve 4200 in an unactuatedstate. Flow channel 4202 and control channel 4204 are formed inelastomeric block 4206 overlying substrate 4205. Flow channel 4202includes a first portion 4202 a and a second portion 4202 b separated byseparating portion 4208. Control channel 4204 overlies separatingportion 4208. As shown in FIG. 21B, in its relaxed, unactuated position,separating portion 4208 remains positioned between flow channel portions4202 a and 4202 b, interrupting flow channel 4202.

FIG. 21C shows a cross-sectional view of valve 4200 wherein separatingportion 4208 is in an actuated position. When the pressure withincontrol channel 4204 is reduced to below the pressure in the flowchannel (for example by vacuum pump), separating portion 4208experiences an actuating force drawing it into control channel 4204. Asa result of this actuation force membrane 4208 projects into controlchannel 4204, thereby removing the obstacle to a flow of materialthrough flow channel 4202 and creating a passageway 4203. Upon elevationof pressure within control channel 4204, separating portion 4208 willassume its natural position, relaxing back into and obstructing flowchannel 4202.

The behavior of the membrane in response to an actuation force may bechanged by varying the width of the overlying control channel.Accordingly, FIGS. 21D-42H show plan and cross-sectional views of analternative embodiment of a normally-closed valve 4201 in which controlchannel 4207 is substantially wider than separating portion 4208. Asshown in cross-sectional views FIGS. 21E-F along line 42E-42E′ of FIG.21D, because a larger area of elastomeric material is required to bemoved during actuation, the actuation force necessary to be applied isreduced.

FIGS. 21G and 21H show a cross-sectional views along line 40G-40G′ ofFIG. 21D. In comparison with the unactuated valve configuration shown inFIG. 21G, FIG. 21H shows that reduced pressure within wider controlchannel 4207 may under certain circumstances have the unwanted effect ofpulling underlying elastomer 4206 away from substrate 4205, therebycreating undesirable void 4212.

Accordingly, FIG. 21I shows a plan view, and FIG. 21J shows across-sectional view along line 21J-21F of FIG. 21I, of valve structure4220 which avoids this problem by featuring control line 4204 with aminimum width except in segment 4204 a overlapping separating portion4208. As shown in FIG. 21J, even under actuated conditions the narrowercross-section of control channel 4204 reduces the attractive force onthe underlying elastomer material 4206, thereby preventing thiselastomer material from being drawn away from substrate 4205 andcreating an undesirable void.

While a normally-closed valve structure actuated in response to pressureis shown in FIGS. 21A-21J, a normally-closed valve in accordance withthe present invention is not limited to this configuration. For example,the separating portion obstructing the flow channel could alternativelybe manipulated by electric or magnetic fields, as described extensivelyabove.

12. Side-Actuated Valve

While the above description has focused upon microfabricated elastomericvalve structures in which a control channel is positioned above andseparated by an intervening elastomeric membrane from an underlying flowchannel, the present invention is not limited to this configuration.FIGS. 22A and 22B show plan views of one embodiment of a side-actuatedvalve structure in accordance with one embodiment of the presentinvention.

FIG. 22A shows side-actuated valve structure 4800 in an unactuatedposition. Flow channel 4802 is formed in elastomeric layer 4804. Controlchannel 4806 abutting flow channel 4802 is also formed in elastomericlayer 4804. Control channel 4806 is separated from flow channel 4802 byelastomeric membrane portion 4808. A second elastomeric layer (notshown) is bonded over bottom elastomeric layer 4804 to enclose flowchannel 4802 and control channel 4806.

FIG. 22B shows side-actuated valve structure 4800 in an actuatedposition. In response to a build up of pressure within control channel4806, membrane 4808 deforms into flow channel 4802, blocking flowchannel 4802. Upon release of pressure within control channel 4806,membrane 4808 would relax back into control channel 4806 and open flowchannel 4802.

While a side-actuated valve structure actuated in response to pressureis shown in FIGS. 22A and 22B, a side-actuated valve in accordance withthe present invention is not limited to this configuration. For example,the elastomeric membrane portion located between the abutting flow andcontrol channels could alternatively be manipulated by electric ormagnetic fields, as described extensively above.

13. Composite Structures

Microfabricated elastomeric structures of the present invention may becombined with non-elastomeric materials to create composite structures.FIG. 23 shows a cross-sectional view of one embodiment of a compositestructure in accordance with the present invention. FIG. 23 showscomposite valve structure 5700 including first, thin elastomer layer5702 overlying semiconductor-type substrate 5704 having channel 5706formed therein. Second, thicker elastomer layer 5708 overlies firstelastomer layer 5702. Actuation of first elastomer layer 5702 to driveit into channel 5706, will cause composite structure 5700 to operate asa valve.

FIG. 24 shows a cross-sectional view of a variation on this theme,wherein thin elastomer layer 5802 is sandwiched between two hard,semiconductor substrates 5804 and 5806, with lower substrate 5804featuring channel 5808. Again, actuation of thin elastomer layer 5802 todrive it into channel 5808 will cause composite structure 5810 tooperate as a valve.

The structures shown in FIG. 23 or 24 may be fabricated utilizing eitherthe multilayer soft lithography or encapsulation techniques describedabove. In the multilayer soft lithography method, the elastomer layer(s)would be formed and then placed over the semiconductor substrate bearingthe channel. In the encapsulation method, the channel would be firstformed in the semiconductor substrate, and then the channel would befilled with a sacrificial material such as photoresist. The elastomerwould then be formed in place over the substrate, with removal of thesacrificial material producing the channel overlaid by the elastomermembrane. As is discussed in detail below in connection with bonding ofelastomer to other types of materials, the encapsulation approach mayresult in a stronger seal between the elastomer membrane component andthe underlying nonelastomer substrate component.

As shown in FIGS. 23 and 24, a composite structure in accordance withembodiments of the present invention may include a hard substrate thatbears a passive feature such as a channels. However, the presentinvention is not limited to this approach, and the underlying hardsubstrate may bear active features that interact with an elastomercomponent bearing a recess. This is shown in FIG. 25, wherein compositestructure 5900 includes elastomer component 5902 containing recess 5904having walls 5906 and ceiling 5908. Ceiling 5908 forms flexible membraneportion 5909. Elastomer component 5902 is sealed against substantiallyplanar nonelastomeric component 5910 that includes active device 5912.Active device 5912 may interact with material present in recess 5904and/or flexible membrane portion 5909.

Many Types of active structures may be present in the nonelastomersubstrate. Active structures that could be present in an underlying hardsubstrate include, but are not limited to, resistors, capacitors,photodiodes, transistors, chemical field effect transistors (chemFET's), amperometric/coulometric electrochemical sensors, fiber optics,fiber optic interconnects, light emitting diodes, laser diodes, verticalcavity surface emitting lasers (VCSEL's), micromirrors, accelerometers,pressure sensors, flow sensors, CMOS imaging arrays, CCD cameras,electronic logic, microprocessors, thermistors, Peltier coolers,waveguides, resistive heaters, chemical sensors, strain gauges,inductors, actuators (including electrostatic, magnetic,electromagnetic, bimetallic, piezoelectric, shape-memory-alloy based,and others), coils, magnets, electromagnets, magnetic sensors (such asthose used in hard drives, superconducting quantum interference devices(SQUIDS) and other types), radio frequency sources and receivers,microwave frequency sources and receivers, sources and receivers forother regions of the electromagnetic spectrum, radioactive particlecounters, and electrometers.

As is well known in the art, a vast variety of technologies can beutilized to fabricate active features in semiconductor and other typesof hard substrates, including but not limited printed circuit board(PCB) technology, CMOS, surface micromachining, bulk micromachining,printable polymer electronics, and TFT and otheramorphous/polycrystalline techniques as are employed to fabricate laptopand flat screen displays.

A variety of approaches can be employed to seal the elastomericstructure against the nonelastomeric substrate, ranging from thecreation of a Van der Waals bond between the elastomeric andnonelastomeric components, to creation of covalent or ionic bondsbetween the elastomeric and nonelastomeric components of the compositestructure. Example approaches to sealing the components together arediscussed below, approximately in order of increasing strength.

A first approach is to rely upon the simple hermetic seal resulting fromVan der Waals bonds formed when a substantially planar elastomer layeris placed into contact with a substantially planar layer of a harder,non-elastomer material. In one embodiment, bonding of RTV elastomer to aglass substrate created a composite structure capable of withstanding upto about 3-4 psi of pressure. This may be sufficient for many potentialapplications.

A second approach is to utilize a liquid layer to assist in bonding. Oneexample of this involves bonding elastomer to a hard glass substrate,wherein a weakly acidic solution (5 μl HCl in H₂O, pH 2) was applied toa glass substrate. The elastomer component was then placed into contactwith the glass substrate, and the composite structure baked at 37° C. toremove the water. This resulted in a bond between elastomer andnon-elastomer able to withstand a pressure of about 20 psi. In thiscase, the acid may neutralize silanol groups present on the glasssurface, permitting the elastomer and non-elastomer to enter into goodVan der Waals contact with each other.

Exposure to ethanol can also cause device components to adhere together.In one embodiment, an RTV elastomer material and a glass substrate werewashed with ethanol and then dried under Nitrogen. The RTV elastomer wasthen placed into contact with the glass and the combination baked for 3hours at 80° C. Optionally, the RTV may also be exposed to a vacuum toremove any air bubbles trapped between the slide and the RTV. Thestrength of the adhesion between elastomer and glass using this methodhas withstood pressures in excess of 35 psi. The adhesion created usingthis method is not permanent, and the elastomer may be peeled off of theglass, washed, and resealed against the glass. This ethanol washingapproach can also be employed used to cause successive layers ofelastomer to bond together with sufficient strength to resist a pressureof 30 psi. In alternative embodiments, chemicals such as other alcoholsor diols could be used to promote adhesion between layers.

An embodiment of a method of promoting adhesion between layers of amicrofabricated structure in accordance with the present inventioncomprises exposing a surface of a first component layer to a chemical,exposing a surface of a second component layer to the chemical, andplacing the surface of the first component layer into contact with thesurface of the second elastomer layer.

A third approach is to create a covalent chemical bond between theelastomer component and functional groups introduced onto the surface ofa nonelastomer component. Examples of derivitization of a nonelastomersubstrate surface to produce such functional groups include exposing aglass substrate to agents such as vinyl silane or aminopropyltriethoxysilane (APTES), which may be useful to allow bonding of the glass tosilicone elastomer and polyurethane elastomer materials, respectively.

A fourth approach is to create a covalent chemical bond between theelastomer component and a functional group native to the surface of thenonelastomer component. For example, RTV elastomer can be created withan excess of vinyl groups on its surface. These vinyl groups can becaused to react with corresponding functional groups present on theexterior of a hard substrate material, for example the Si—H bondsprevalent on the surface of a single crystal silicon substrate afterremoval of native oxide by etching. In this example, the strength of thebond created between the elastomer component and the nonelastomercomponent has been observed to exceed the materials strength of theelastomer components.

14. Cell Pen

In yet a further application of the present invention, an elastomericstructure can be utilized to manipulate organisms or other biologicalmaterial. FIGS. 26A-D show plan views of one embodiment of a cell penstructure in accordance with the present invention.

Cell pen array 4400 features an array of orthogonally-oriented flowchannels 4402, with an enlarged “pen” structure 4404 at the intersectionof alternating flow channels. Valve 4406 is positioned at the entranceand exit of each pen structure 4404. Peristaltic pump structures 4408are positioned on each horizontal flow channel and on the vertical flowchannels lacking a cell pen structure.

Cell pen array 4400 of FIG. 26A has been loaded with cells A-H that havebeen previously sorted. FIGS. 26B-C show the accessing and removal ofindividually stored cell C by 1) opening valves 4406 on either side ofadjacent pens 4404 a and 4404 b, 2) pumping horizontal flow channel 4402a to displace cells C and G, and then 3) pumping vertical flow channel4402 b to remove cell C. FIG. 26D shows that second cell G is moved backinto its prior position in cell pen array 4400 by reversing thedirection of liquid flow through horizontal flow channel 4402 a.

The cross-flow channel architecture illustrated shown in FIGS. 26A-D canbe used to perform functions other than the cell pen just described. Forexample, the cross-flow channel architecture can be utilized in mixingapplications.

This is shown in FIGS. 28A-B, which illustrate a plan view of mixingsteps performed by a microfabricated structure in accordance anotherembodiment of the present invention. Specifically, portion 7400 of amicrofabricated mixing structure comprises first flow channel 7402orthogonal to and intersecting with second flow channel 7404. Controlchannels 7406 overlie flow channels 7402 and 7404 and form valve pairs7408 a-b and 7408 c-d that surround each intersection 7412.

As shown in FIG. 28A, valve pair 7408 a-b is initially opened whilevalve pair 7408 c-d is closed, and fluid sample 7410 is flowed tointersection 7412 through flow channel 7402. Valve pair 7408 c-d is thenactuated, trapping fluid sample 7410 at intersection 7412.

Next, as shown in FIG. 28B, valve pairs 7408 a-b and 7408 c-d areopened, such that fluid sample 7410 is injected from intersection 7412into flow channel 7404 bearing a cross-flow of fluid. The process shownin FIGS. 28A-B can be repeated to accurately dispense any number offluid samples down cross-flow channel 7404. FIG. 54 plots Log(R/B) vs.number of slugs injected for one embodiment of a cross-flow injectionsystem in accordance with the present invention. The reproducibility andrelative independence of metering by cross-flow injection from processparameters such as flow resistance is further evidenced by FIG. 54,which plots injected volume versus number of injection cycles forcross-channel flow injection under a variety of flow conditions. FIG. 54shows that volumes metered by cross-flow injection techniques increaseon a linear basis over a succession of injection cycles. This linearrelationship between volume and number of injection cycles is relativelyindependent of flow resistance parameters such as elevated fluidviscosity (imparted by adding 25% glycerol) and the length of the flowchannel (1.0-2.5 cm).

While the embodiment shown and described above in connection with FIGS.28A-B utilizes linked valve pairs on opposite sides of the flow channelintersections, this is not required by the present invention. Otherconfigurations, including linking of adjacent valves of an intersection,or independent actuation of each valve surrounding an intersection, arepossible to provide the desired flow characteristics. With theindependent valve actuation approach however, it should be recognizedthat separate control structures would be utilized for each valve,complicating device layout.

15. Metering by Volume Exclusion

Many high throughput screening and diagnostic applications call foraccurate combination and of different reagents in a reaction chamber.Given that it is frequently necessary to prime the channels of amicrofluidic device in order to ensure fluid flow, it may be difficultto ensure mixed solutions do not become diluted or contaminated by thecontents of the reaction chamber prior to sample introduction.

Volume exclusion is one technique enabling precise metering of theintroduction of fluids into a reaction chamber. In this approach, areaction chamber may be completely or partially emptied prior to sampleinjection. This method reduces contamination from residual contents ofthe chamber contents, and may be used to accurately meter theintroduction of solutions in a reaction chamber.

Specifically, FIGS. 29A-D show cross-sectional views of a reactionchamber in which volume exclusion is employed to meter reactants. FIG.29A shows a cross-sectional view of portion 6300 of a microfluidicdevice comprising first elastomer layer 6302 overlying second elastomerlayer 6304. First elastomer layer 6302 includes control chamber 6306 influid communication with a control channel (not shown). Control chamber6306 overlies and is separated from dead-end reaction chamber 6308 ofsecond elastomer layer 6304 by membrane 6310. Second elastomer layer6304 further comprises flow channel 6312 leading to dead-end reactionchamber 6308.

FIG. 29B shows the result of a pressure increase within control chamber6306. Specifically, increased control chamber pressure causes membrane6310 to flex downward into reaction chamber 6308, reducing by volume Vthe effective volume of reaction chamber 6308. This in turn excludes anequivalent volume V of reactant from reaction chamber 6308, such thatvolume V of first reactant X is output from flow channel 6312. The exactcorrelation between a pressure increase in control chamber 6306 and thevolume of material output from flow channel 6312 can be preciselycalibrated.

As shown in FIG. 29C, while elevated pressure is maintained withincontrol chamber 6306, volume V′ of second reactant Y is placed intocontact with flow channel 6312 and reaction chamber 6308.

In the next step shown in FIG. 29D, pressure within control chamber 6306is reduced to original levels. As a result, membrane 6310 relaxes andthe effective volume of reaction chamber 6308 increases. Volume V ofsecond reactant Y is sucked into the device. By varying the relativesize of the reaction and control chambers, it is possible to accuratelymix solutions at a specified relative concentration. It is worth notingthat the amount of the second reactant Y that is sucked into the deviceis solely dependent upon the excluded volume V, and is independent ofvolume V′ of Y made available at the opening of the flow channel.

While FIGS. 29A-D show a simple embodiment of the present inventioninvolving a single reaction chamber, in more complex embodimentsparallel structures of hundreds or thousands of reaction chambers couldbe actuated by a pressure increase in one control line.

Moreover, while the above description illustrates two reactants beingcombined at a relative concentration that fixed by the size of thecontrol and reaction chambers, a volume exclusion technique could beemployed to combine several reagents at variable concentrations in asingle reaction chamber. One possible approach is to use several,separately addressable control chambers above each reaction chamber. Anexample of this architecture would be to have ten separate control linesinstead of a single control chamber, allowing ten equivalent volumes tobe pushed out or sucked in.

Another possible approach would utilize a single control chamberoverlying the entire reaction chamber, with the effective volume of thereaction chamber modulated by varying the control chamber pressure. Inthis manner, analog control over the effective volume of the reactionchamber is possible. Analog volume control would in turn permit thecombination of many solutions reactants at arbitrary relativeconcentrations.

An embodiment of a method of metering a volume of fluid in accordancewith the present invention comprises providing a chamber having a volumein an elastomeric block separated from a control recess by anelastomeric membrane, and supplying a pressure to the control recesssuch that the membrane is deflected into the chamber and the volume isreduced by a calibrated amount, excluding from the chamber thecalibrated volume of fluid.

II. Microfluidic Free Interface Diffusion Techniques

A microfluidic free interface (μFI) in accordance with embodiments ofthe present invention is a localized interface between at least onestatic fluid and another fluid wherein mixing between them is dominatedby diffusion rather than by convective flow. For the purposes of thisapplication, the term “fluid” refers to a material having a viscositybelow a particular maximum. Examples of such maximum viscosities includebut are not limited to 1000 CPoise, 900 CPoise, 800 CPoise, 700 CPoise,600 CPoise, 500 CPoise, 400 CPoise, 300 CPoise, 250 CPoise, and 100CPoise, and therefore exclude gels or polymers containing materialstrapped therein.

In a microfluidic free interface in accordance with an embodiment of thepresent invention, at least one dimension of the interface is restrictedin magnitude such that viscous forces dominate other forces. Forexample, in a microfluidic free interface in accordance with anembodiment of the present invention, the dominant forces acting upon thefluids are viscous rather than buoyant, and hence the microfluidic freeinterface may be characterized by an extremely low Grashof number (seediscussion below). The microfluidic free interface may also becharacterized by its localized nature relative to the total volumes ofthe fluids, such that the volumes of fluid exposed to the steeptransient concentration gradients present initially after formation ofthe interface between the pure fluids is limited.

The properties of a microfluidic free interface created in accordancewith embodiments of the present invention may be contrasted with anon-free microfluidic interface, as illustrated in FIGS. 33A and 33B.Specifically, FIG. 33A shows a simplified cross-sectional view of amicrofluidic free interface in accordance with an embodiment of thepresent invention. Microfluidic free interface 7500 of FIG. 33A isformed between first fluid A and second fluid B present within channel7502. The free microfluidic interface 7500 is substantially linear, withthe result that the steep concentration gradient arising between fluidsA and B is highly localized within the channel.

As described above, the dimensions of channel 7502 are extremely small,with the result that non-slip layers immediately adjacent to the wallsof the channel in fact occupy most of the volume of the channel. As aresult, viscosity forces are much greater than buoyant forces, andmixing between fluids A and B along interface 7500 occurs almostentirely as a result of diffusion, with little or no convective mixing.Conditions associated with the microfluidic free interface ofembodiments of the present invention can be expressed in terms of theGrashof number (Gr) per Equation (2) below, an expression of therelative magnitude of buoyant and viscous forces:

Gr∝B/V,  (2)

where:

-   -   Gr=Grashof number;    -   B=buoyancy force; and    -   V=viscous force.        Microfluidic free interfaces in accordance with embodiments of        the present invention would be expected to exhibit a Grashof        number of 1 or less. The Grashof number expected with two fluids        having the same density is zero, and thus Grashof, numbers very        close to zero would be expected to be attained.

The embodiment of a microfluidic free interface illustrated above inFIG. 33A may be contrasted with the conventional non-microfluidic freeinterface shown in FIG. 33B. Specifically, first and second fluids A andB are separated by an interface 7504 that is not uniform or limited bythe cross-sectional width of channel 7502. The steep concentrationgradient occurring at the interface is not localized, but is insteadpresent at various points along the length of the channel, exposingcorrespondingly large volumes of the fluids to the steep gradients. Inaddition, viscosity forces do not necessarily dominate over buoyancyforces, with the result that mixing of fluids A and B across interface7504 can occur both as the result of diffusion and of convective flow.The Grashof number exhibited by such a conventional non-microfluidicinterface would be expected to exceed 1.

TABLE A below provides a summary of the properties of a freemicrofluidic interface in accordance with an embodiment of the presentinvention, as contrasted with a non-microfluidic free interface.

TABLE A PROPERTY μFI non-μFI fluid forces dominated by a combinationviscous force of viscous and buoyant forces Grashof No. low (i.e. <1)higher (i.e. >1) mechanism for mixing predominantly a combination offluids across diffusion of diffusion and interface convective flowlocation of steepest localized non-localized concentration gradientbetween the fluids in their pure form approximate magnitude 100 nm-100μm >100 μm of restrictive dimen- sion at interface interface shapeplanar non-planar

1. Creation of Microfluidic Free Interface

A microfluidic free interface in accordance with embodiments of thepresent invention may be created in a variety of ways. One approach isto utilize the microfabricated elastomer structures previouslydescribed. Specifically, in certain embodiments the elastomeric materialfrom which microfluidic structures are formed is relatively permeable tocertain gases. This gas permeability property may be exploited utilizingthe technique of pressurized out-gas priming (POP) to form well-defined,reproducible fluidic interfaces.

FIG. 34A shows a plan view of a flow channel 9600 of a microfluidicdevice in accordance with an embodiment of the present invention. Flowchannel 9600 is separated into two halves by actuated valve 9602. Priorto the introduction of material, flow channel 9600 contains a gas 9604.

FIG. 34B shows the introduction of a first solution 9606 to first flowchannel portion 9600 a under pressure, and the introduction of a secondsolution 9608 to second flow channel portion 9600 b under pressure.Because of the gas permeability of the surrounding elastomer material9607, gas 9604 is displaced by the incoming solutions 9608 and 9610 andout-gasses through elastomer 9607.

As shown in FIG. 34C, the pressurized out-gas priming of flow channelportions 9600 a and 9600 b allows uniform filling of these dead-endedflow channel portions without air bubbles. Upon deactuation of valve9602 as shown in FIG. 34D, microfluidic free interface 9612 is defined,allowing for formation of a diffusion gradient between the fluids.

While the specific embodiment just described exploits the permeabilityof the bulk material to dead end fill two or more chambers or channelsseparated by a closed valve, and creates a microfluidic free interfacebetween the static fluids by the subsequent opening of this valve, othermechanisms for realizing a microfluidic free interface are possible.

For example, FIG. 47 shows one potential alternative method forestablishing a microfluidic free interface diffusion in accordance withthe present invention. Microfluidic channel 8100 carries fluid Aexperiencing a convective flow in the direction indicated by the arrow,such that static non-slip layers 8102 are created along the walls offlow channel 8100. Branch channel 8104 and dead-ended chamber 8106contain static fluid B. Because material surrounding the dead-endedchannel and chamber provide a back pressure, fluid B remains static andmicrofluidic free interface 8110 is created at mouth 8112 of branchchannel 8108 between flowing fluid A and static fluid B. As describedbelow, diffusion of fluid A or components thereof across themicrofluidic free interface can be exploited to obtain useful results.While the embodiment shown in FIG. 47 includes a dead-ended branchchannel and chamber, this is not required by the present invention, andthe branch channel could connect with another portion of the device, aslong as a sufficient counter pressure was maintained to prevent any netflow of fluid through the channel.

Another potential alternative method for establishing a microfluidicfree interface diffusion assay is the use of break-through valves andchambers. A break-through valve is not a true closing valve, but rathera structure that uses the surface tension of the working fluid to stopthe advance of the fluid. Since these valves depend on the surfacetension of the fluid they can only work while a free surface exists atthe valve; not when the fluid continuously fills both sides and theinterior of the valve structure.

A non-exclusive list of ways to achieve such a valve include but are notlimited to patches of hydrophobic material, hydrophobic treatment ofcertain areas, geometric constrictions (both in height and width) of achannel, geometric expansions (both in height and in width of achannel), changes in surface roughness on walls of a channel, andapplied electric potentials on the walls.

These “break-through” valves may be designed to withstand a fixed andwell defined pressure before they “break through” and allow fluid topass nearly unimpeded. The pressure in the channel can be controlled andhence the fluid can be caused to advance when desired. Different methodsof controlling this pressure include but are not limited to externallyapplied pressure at an input or output port, pressure derived fromcentrifugal force (i.e. by spinning the device), pressure derived fromlinear acceleration (i.e. applying an acceleration to the device with acomponent parallel to the channel), electrokinetic pressure, internallygenerated pressure from bubble formation (by chemical reaction or byhydrolysis), pressure derived from mechanical pumping, or osmoticpressure.

“Break-through” valves may be used to create a microfluidic freeinterface as shown and described in connection with FIGS. 35A-E. FIG.35A shows a simplified plan view of a device for creating a microfluidicfree interface utilizing break through valves. First chamber 9100 is influid communication with second chamber 9102 through branches 9104 a and9104 b respectively, of T-shaped channel 9104.

First break through valve 9106 is located at outlet 9108 of firstchamber 9100. Second break through valve 9110 is located in branch 9104b upstream of inlet 9105 of second chamber 9102. Third break throughvalve 9112 is located at outlet 9114 of second chamber 9102.Breakthrough valves 9106, 9110, and 9112 may be formed from hydrophobicpatches, a constriction in the width of the flow channel, or some otherway as described generally above. In FIGS. 35A-E, an open break throughvalve is depicted as an unshaded circle, and a closed break throughvalve is depicted as a shaded circle.

In the initial stage shown in FIG. 35B, first chamber 9100 is chargedwith first fluid 9116 introduced through stem 9104 c and branch 9104 aof T-shaped channel 9104 and chamber inlet 9107 at a pressure below thebreak through pressure of any of the valves 9106, 9110, and 9112. In thesecond stage shown in FIG. 35C, second chamber 9102 is charged with abuffer or other intermediate fluid 9118 introduced through stem 9104 cof T-shaped channel 9104 and inlet 9105 at a pressure below the breakthrough pressure of valve 9106 but greater than the break throughpressures of valves 9110 and 9112.

In the third stage shown in FIG. 35D, intermediate fluid 9118 isreplaced in second chamber 9102 with second fluid 9120 introducedthrough stem 9104 c of T-shaped channel 9104 and inlet 9105 at apressure below the break through pressure of valve 9106 but greater thanthe break through pressures of valves 9110 and 9112. In the final stagedepicted in FIG. 35E, second fluid 9120 has replaced the intermediatefluid, leaving the first and second fluids 9116 and 9120 in separatechambers but fluidically connected through T-junction 9104, creating amicrofluidic free interface 9122.

The use of break through valves to create a microfluidic free interfacein accordance with embodiments of the present invention is not limitedto the specific example given above. For example, in alternativeembodiments the step of flushing with a buffer or intermediate solutionis not required, and the first solution could be removed by flushingdirectly with the second solution, with potential unwanted by-productsof mixing removed by the initial flow of the second solution through thechannels and chambers.

While the embodiments just described create the microfluidic freeinterface in a closed microfluidic device, this is not required byembodiments in accordance with the present invention. For example, analternative embodiment in accordance with the present invention mayutilize capillary forces to connect two reservoirs of fluid. In oneapproach, the open wells of a micro-titer plate could be connected by asegment of a glass capillary. The first solution would be dispensed intoone well such that it fills the well and is in contact with the glasscapillary. Capillary forces cause the first solution to enter and flowto the end of the capillary. Once at the end, the fluid motion ceases.Next, the second solution is added to the second well. This solution isin contact with the first solution at the capillary inlet and creates amicrofluidic interface between the two wells at the end of thecapillary.

The connecting path between the two wells need not be a glass capillary,and in alternative embodiments could instead comprise a strip ofhydrophilic material, for example a strip of glass or a line of silicadeposited by conventional CVD or PVD techniques. Alternatively, theconnecting paths could be established by paths of less hydrophobicmaterial between patterned regions of highly hydrophobic material.Moreover, there could be a plurality of such connections between thewells, or a plurality of interconnected chambers in variousconfigurations. Such interconnections could be established by the userprior to use of the device, allowing for rapid and efficient variationin fluidic conditions.

Where as in the previous example the two reservoirs are not enclosed bya microfluidic device but are connected instead through a microfluidicpath, an alternative embodiment could have reservoirs both enclosed andnot enclosed. For example, sample could be loaded into a microfluidicdevice and pushed to the end of an exit capillary or orifice (by any ofthe pressure methods described above). Once at the end of the exitcapillary, the capillary could be immersed in a reservoir of reagent. Inthis way, the microfluidic free interface is created between theexternal reservoir and the reservoir of reagent in the chip. This methodcould be used in parallel with many different output capillaries ororifices to screen a single sample against a plurality of differentreagents using microfluidic free interface diffusion.

In the example just described, the reagent is delivered from one or manyinlets to one or many different outlets “through” a microfluidic device.Alternatively, this reagent can be introduced through the same orificethat is to be used to create the microfluidic interface. Thesample-containing solution could be aspirated into a capillary (eitherby applying suction, or by capillary forces, or by applying pressure tothe solution) and then the capillary may be immersed in a reservoir ofcounter-reagent, creating a microfluidic interface between the end ofthe capillary and the reservoir. This could be done in a large array ofcapillaries for the parallel screening of many different reagents. Verysmall volumes of sample could be used since the capillaries can have afixed length beyond which the sample will not advance. Forcrystallization applications (see below), the capillaries could beremoved and mounted in an x-ray beam for diffraction studies, withoutrequiring handling of the crystals.

2. Reproducible Control Over Equilibration Parameters

One advantage of the use of microfluidic free interface diffusion inaccordance with embodiments of the present invention is the ability tocreate uniform and continuous concentration gradients that reproduciblysample a wide range of conditions. As the fluids on either side of theinterface diffuse into one another, a gradient is established along thediffusion path, and a continuum of conditions is simultaneously sampled.Since there is a variation in the conditions, both in space and time,information regarding the location and time of positive results (i.e.crystal formation) may be used in further optimization.

In many applications it is desirable to create a gradient of a conditionsuch as pH, concentration, or temperature. Such gradients may be usedfor screening applications, optimization of reaction conditions,kinetics studies, determination of binding affinities, dissociationconstants, enzyme-rate profiling, separation of macromolecules, and manyother applications. Due principally to the suppression of convectiveflow, diffusion across a microfluidic free interface in accordance withan embodiment of the present invention may be used to establish reliableand well-defined gradient.

The dimensional Einstein equation (3) may be employed to obtain a roughestimate of diffusion times across a microfluidic free interface.

$\begin{matrix}{{t = \frac{x^{2}}{4D}};} & (3)\end{matrix}$

where:

-   -   t=diffusion time;    -   x=longest diffusion length; and    -   D=diffusion coefficient        Generally, as shown in Equation (4) below, the diffusion        coefficient varies inversely with the radius of gyration, and        therefore as one over the cube root of molecular weight.

$\begin{matrix}{{D \propto \frac{1}{r} \propto \frac{1}{m^{1/3}}};} & (4)\end{matrix}$

where:

-   -   D=diffusion coefficient;    -   r=radius of gyration; and    -   m=molecular weight        In reviewing equation (4), it is important to recognize that        correlation between the radius of gyration (r) and the molecular        weight (m) is only an approximation. Because of the dominance of        viscous forces over inertial forces, the diffusion coefficient        is in fact independent of molecular weight and is instead        dependent upon the size and hence drag experienced by the        diffusing particle.

As compared with the rough 1.5 hr equilibration time for a dye, anapproximate equilibration time for a protein of 20 KDa over the samedistance is estimated to be approximately 45 hours. The equilibrationtime for a small salt of a molecular weight of 100 Da over the samedistance is about 45 minutes.

FIGS. 36A-D are simplified schematic diagrams plotting concentrationversus distance for a solution A and a solution B in contact along afree interface. FIGS. 36A-D show that over time, a continuous and broadrange of concentration profiles of the two solutions is ultimatelycreated.

Moreover, varying the equilibration rate by changing the geometry overtime of connecting channels may be used on a single device to explorethe effect of equilibration dynamics. FIGS. 37A-D show an embodiment inwhich a gradient of concentrations, initially established by the partialdiffusive equilibration of two solutions from a micro-free interface,can be captured by actuation of containment valves.

FIG. 37A shows flow channel 10000 that is overlapped at intervals by aforked control channel 10002 to define a plurality of chambers (A-G)positioned on either side of a separately-actuated interface valve10004. FIG. 37B plots solvent concentration at an initial time, wheninterface valve 10004 is actuated and a first half 10000 a of the flowchannel has been mixed with a first solution, and a second half 10000 bof the flow channel has been primed with a second solution. FIG. 37Cplots solvent concentration at a subsequent time T₁, when controlchannel 10002 is actuated to define the seven chambers (A-G), whichcapture the concentration gradient at that particular point in time.FIG. 37D plots relative concentration of the chambers (A-G) at time T₁.

In the embodiment shown in FIG. 37A, actuation of the forked controlchannel simultaneously creates the plurality of chambers A-G. However,this is not required, and in alternative embodiments of the presentinvention multiple control channels could be utilized to allowindependent creation of chambers A-G at different time intervals,thereby allow additional diffusion to occur after an initial set ofchambers are created immediately adjacent to the free interface.

The relative concentrations resulting from diffusion across a fluidicinterface is determined not only by thermodynamic conditions exploredduring the equilibration, but also by the rate at which equilibrationtakes place. It is therefore potentially valuable to control thedynamics of equilibration.

In conventional macroscopic diffusion methods, only coarse control overthe dynamics of equilibration may be available through manipulation ofinitial conditions. For macroscopic free interface diffusion, oncediffusion begins, the experimenter has no control over the subsequentequilibration rate. For hanging drop experiments, the equilibration ratemay be changed by modifying the size of the initial drop, the total sizeof the reservoir, or the temperature of incubation. In microbatchexperiments, the rate at which the sample is concentrated may be variedby manipulating the size of the drop, and the identity and amount of thesurrounding oil. Since the equilibration rates depend in a complicatedmanner on these parameters, the dynamics of equilibration may only bechanged in a coarse manner. Moreover, once the experiment has begun, nofurther control over the equilibration dynamics is available.

By contrast, in a fluidic free interface experiment in accordance withan embodiment of the present invention, the parameters of diffusiveequilibration rate may also be controlled by manipulating dimensions ofchambers and connecting channels of a microfluidic structure. Forexample, in a microfluidic structure comprising reservoirs in fluidcommunication through a constricted channel, where no appreciablegradient exists in the reservoirs due to high concentrations orreplenishment of material, to good approximation the time required forequilibration varies linearly with the required diffusion length. Theequilibration rate also depends on the cross-sectional area of theconnecting channels. The required time for equilibration may thereforebe controlled by changing both the length, and the cross-sectional areaof the connecting channels.

For example, FIG. 40A shows a plan view of a simple embodiment of amicrofluidic structure in accordance with the present invention.Microfluidic structure 9701 comprises reservoirs 9700 and 9702containing first fluid A and second fluid B, respectively. Reservoirs9700 and 9702 are connected by channel 9704. Valve 9706 is positioned onthe connecting channel between reservoirs 9700 and 9702.

Connecting channel 9704 has a much smaller cross-sectional area thaneither of the reservoirs. For example, in particular embodiments ofmicrofluidic structures in accordance with the present invention, theratio of reservoir/channel cross-sectional area and thus the ratio ofmaximum ratio of cross-sectional area separating the two fluids, mayfall between 500 and 25,000. The minimum of this range describes a50×50×50 μm chamber connected to a 50×10 μm channel, and the maximum ofthis range describes a 500×500×500 μm chamber connected to a 10×1 μmchannel.

Initially, reservoirs 9700 and 9702 are filled with respective fluids,and valve 9706 is closed. Upon opening valve 9706, a microfluidic freeinterface in accordance with an embodiment of the present invention iscreated, and fluids A and B diffuse across this interface through thechannel into the respective reservoirs. Moreover, where the amount ofdiffusing material present in one reservoir is large and the capacity ofthe other reservoir to receive material without undergoing a significantconcentration change is also large, the concentrations of material inthe reservoirs will not change appreciably over time, and a steady stateof diffusion will be established.

Diffusion of fluids in the simple microfluidic structure shown in FIG.40A may be described by relatively simple equations. For example, thenet flux of a chemical species from one chamber to the other may besimply described by equation (5):

$\begin{matrix}{{J = {D*A*\frac{\Delta \; C}{L}}};} & (5)\end{matrix}$

where:

-   -   J=net flux of chemical species    -   D=diffusion constant of the chemical species;    -   A=cross-sectional area of the connecting channel;    -   ΔC=concentration difference between the two channels; and    -   L=length of the connection channel.

Following integration and extensive manipulation of the terms ofequation (5), the characteristic time t for the equilibration of the twochambers, where one volume V₁ is originally at concentration C and theother volume V₂ is originally at concentration 0, can therefore be takento be as shown in Equation (6) below:

$\begin{matrix}{{\tau = {\frac{1}{{V_{1}/V_{2}} + 1}*\frac{1}{D}*\frac{L}{A/V_{1}}}};} & (6)\end{matrix}$

where:

-   -   τ=equilibration time;    -   V₁=volume of chamber initially containing the chemical species;    -   V₂=volume of chamber into which the chemical species is        diffusing;    -   D=diffusion constant of the chemical species;    -   A=cross-sectional area of the connecting channel; and    -   L=length of the connection channel.

Therefore, for a given initial concentration of a chemical species in achamber of a defined volume, the characteristic equilibration timedepends in a linear manner from the diffusive length L and the ratio ofthe cross-sectional area to the volume (hereafter referred to simply asthe “area”), with the understanding that the term “area” refers to thearea normalized by the volume of the relevant chamber. Where twochambers are connected by a constricted channel, as in the structure ofFIG. 40A, the concentration drop from one channel to the other occursprimarily along the connecting channel and there is no appreciablegradient present in the chamber. This is shown in FIG. 40B, which is asimplified plot of concentration versus distance for the structure ofFIG. 40A.

The behavior of diffusion between the chambers of the microfluidicstructure of FIG. 40A can be modeled, for example, utilizing the PDEtoolbox of the MATLAB® software program sold by The MathWorks Inc. ofNatick, Mass. FIGS. 41 and 42 accordingly show the results of simulatingdiffusion of sodium chloride from a 300 um×300 um×100 um chamber toanother chamber of equal dimensions, through a 300 um long channel witha cross-sectional area of 1000 um. The initial concentrations of thechambers are 1 M and 0 M, respectively.

FIG. 41 plots the time required for the concentration in one of thereservoirs to reach 0.6 of the final equilibration concentration, versuschannel length. FIG. 41 shows the linear relationship between diffusiontime and channel length for this simple microfluidic system.

FIG. 42 plots the inverse of the time required for the concentration inone of the reservoirs to reach 0.6 of the final equilibrationconcentration (T_(0.6)), versus the area of the fluidic interfacecreated upon opening of the valve. FIG. 42 shows the linear relationshipbetween these parameters. The simple relationship between theequilibration time constant and the parameters of channel length and1/channel area allows for a reliable and intuitive method forcontrolling the rate of diffusive mixing across a microfluidic freeinterface in accordance with an embodiment of the present invention.

This relationship further allows for one reagent to be diffusively mixedwith a plurality of others at different rates that may be controlled bythe connecting channel geometry. For example, FIG. 38A shows three setsof pairs of compound chambers 9800, 9802, and 9804, each pair connectedby microchannels 9806 of a different length Δx. FIG. 38B plotsequilibration time versus equilibration distance. FIG. 38B shows thatthe required time for equilibration of the chambers of FIG. 38A variesas the length of the connecting channels.

FIG. 39 shows four compound chambers 9900, 9902, 9904, and 9906, eachhaving different arrangements of connecting microchannel(s) 9908.Microchannels 9908 have the same length, but differ in cross-sectionalarea and/or number of connecting channels. The rate of equilibration maythus be increased/decreased by decreasing/increasing the cross-sectionalarea, for example by decreasing/increasing the number of connectingchannels or the dimensions of those channels.

Another desirable aspect of microfluidic free interface diffusionstudies in accordance with embodiments of the present invention is theability to reproducibly explore a wide range of phase space. Forexample, it may be difficult to determine, a priori, which thermodynamicconditions will be favorable for a particular application (i.e.nucleation/growth of protein crystals), and therefore it is desirablethat a screening method sample as much of phase-space (as manyconditions) as possible. This can be accomplished by conducting aplurality of assays, and also through the phase space sampled during theevolution of each assay in time.

FIG. 43 shows the results of simulating the counter-diffusion oflysosyme and sodium chloride utilizing the microfluidic structure shownin FIG. 40A, with different relative volumes of the two reservoirs, andwith initial concentrations normalized to 1. FIG. 43 presents a phasediagram depicting the phase space between fluids A and B, and the pathin phase space traversed in the reservoirs as the fluids diffuse acrossthe microfluidic free interface created by the opening of the valve inFIG. 40A. FIG. 43 shows that the phase space sampled depends upon theinitial relative volumes of the fluids contained in the two reservoirs.By utilizing arrays of chamber with different sample volumes, and thenidentifying instances where diffusion across the channel yieldeddesirable results (i.e. crystal formation), promising starting pointsfor additional experimentation can be determined.

As described above, varying the length or cross-sectional area of achannel connecting two reservoirs changes the rate at which the speciesare mixed. However, so long as the channel volume remains small comparedas compared with the total reaction volume, there is little or no effecton the evolution of concentration in the chambers through phase space.The kinetics of the mixing are therefore decoupled from the phase-spaceevolution of the reaction, allowing the exercise independent controlover the kinetic and thermodynamic behavior of the diffusion.

For example, it is often desirable in crystallography to slow down theequilibration so as to allow for the growth of fewer and higher qualitycrystals. In conventional techniques this is often attempted by addingnew chemical constituents such as glycerol, or by using microbatchmethods. However, this addition of constituents is not wellcharacterized, is not always effective, and may inhibits the formationof crystals. Microbatch methods also may pose the disadvantage oflacking a driving force to promote continued crystal growth as proteinin the solution surrounding the crystal is depleted. Through the use ofdiffusion across a microfluidic free interface in accordance with anembodiment of the present invention, crystal formation may be slowed bya well-defined amount without altering the phase-space evolution, simplyby varying the width or cross-sectional area of the connecting channel.

The ability to control the rate at which equilibration proceeds hasfurther consequences in cases were one wishes to increase the totalvolume of a reaction while conserving both the thermodynamics and themicrofluidic free interface diffusion mixing. One such case arises againin the context of protein crystallography, in which an initial, smallvolume crystallization assay results in crystals of insufficient sizefor diffraction studies. In such a case, it is desirable to increase thereaction volume and thereby provide more protein available for crystalgrowth, while at the same time maintaining the same diffusive mixing andpath through phase space. By increasing the chamber volumesproportionally and decreasing the area of the channel, the area of theinterface relative to the total assay volume is reduced, and a largervolume would pass through the same phase space as in the original smallvolume conditions.

While the above description has focused upon diffusion of a singlespecies, gradients of two or more of species which do not interact witheach other may be created simultaneously and superimposed to create anarray of concentration conditions. FIG. 52 shows a plan view of oneexample of a microfluidic structure for creating such superimposedgradients. Flat, shallow chamber 8600 constricted in the verticaldirection is connected at its periphery to reservoirs 8602 and 8604having fixed concentrations of chemical species A and B, respectively.Sink 8606 in the form of a reservoir is maintained at a substantiallylower concentration of species A and B. After the initial transientequilibration, stable and well-defined gradients 8608 and 8610 ofspecies A and B respectively, are established in two dimensions.

As evident from inspection of FIG. 52, the precise shape and profile ofthe concentration gradient will vary according to a host of factors,including but not limited to the relative location and number of inletsto the chamber, which can also act as concentration sinks for thechemical species not contained therein (i.e. reservoir 8604 may act as asink for chemical species A). However, the spatial concentrationprofiles of each chemical species within the chamber may readily bemodeled using the MATLAB program previously described to describe atwo-dimensional, well-defined, and continuous spatial gradient.

The specific embodiment illustrated in FIG. 52 offers the disadvantageof continuous diffusion of materials. Hence, where diffusion of productsof reaction between the diffusing species is sought to be discerned,these products will themselves diffuse in the continuous gradient,thereby complicating analysis.

Accordingly, FIG. 53 shows a simplified plan view of an alternativeembodiment of a microfluidic structure for accomplishing diffusion intwo dimensions. Grid 8700 of intersecting orthogonal channels 8702establishes a spatial concentration gradient. Reservoirs 8704 and 8708of fixed concentrations of chemical species A and B are positioned onadjacent edges of grid 8700. Opposite to these reservoirs on the gridare two sinks 8703 of lower concentration of the chemical present in theopposing reservoirs.

Surrounding each channel junction 8710 are two pairs of valves 8712 and8714 which control diffusion through the grid in the vertical andhorizontal directions, respectively. Initially, only valve pairs 8714are opened to create a well-defined diffusion gradient of the firstchemical in the horizontal direction. Next, valve pairs 8714 are closedand valve pairs 8712 opened to create a well-defined diffusion gradientof the second chemical in the vertical direction. Isolated by adjacenthorizontal valves, the gradient of the first chemical species remainspresent in regions between the junctions.

Once the second (vertical) gradient is established, the two gradientscan be combined and by opening all the valve pairs for a short time toallow partial diffusive equilibration. After the period of diffusion haspassed, all the valve pairs are closed to contain the superimposedgradient. Alternatively, valve pairs 8712 and 8714 can be closed to haltdiffusion in the vertical direction, with every second horizontal valveopened to create separate isolated chambers.

3. Control Over Environmental Factors

The above discussion has focused upon altering the chemical environmentthrough diffusive mixing across a microfluidic free interface betweentwo fluids. Other factors, however, may also influence interactionbetween the fluids on either side of the microfluidic free interface.Such additional factors include, but are not limited to, temperature,pressure, and the concentration of materials in the fluids.

In specific embodiments in accordance with the present invention,control over temperature during diffusive mixing may be accomplishedutilizing a composite elastomer/silicon structure previously described.Specifically, a Peltier temperature control structure may be fabricatedin an underlying silicon substrate, with the elastomer aligned to thesilicon such that a crystallization chamber is proximate to the Peltierdevice. Application of voltage of an appropriate polarity and magnitudeto the Peltier device may control the temperature of fluids within achamber or channel.

Alternatively, as described by Wu et al. in “MEMS Flow Sensors forNano-fluidic Applications”, Sensors and Actuators A 89 152-158 (2001),microfabricated chambers or channels could be heated and cooled throughthe selective application of current to a micromachined resistorstructure resulting in ohmic heating. Moreover, the temperature of thefluid could be detected by monitoring the resistance of the heater overtime. The Wu et al. paper is hereby incorporated by reference for allpurposes.

It may also be useful to establish a temperature gradient across amicrofabricated free interface in accordance with the present invention.Such a temperature gradient would subject the diffusing fluids to abroad spectrum of temperatures during mixing, allowing for extremelyprecise determination of optimum temperatures.

With regard to controlling pressure during diffusive mixing, embodimentsof the present invention which meter fluids by volume exclusion areparticularly advantageous. Specifically, once the chamber has beencharged with appropriate fluid volumes, a chamber inlet valve may bemaintained shut while the membrane overlying the chamber is actuated,thereby causing pressure to increase in the chamber. Structures inaccordance with the present invention employing techniques other thanvolume exclusion could exert pressure control by including flow channelsand associated membranes adjacent to the diffusion channel or chamberthat are specifically relegated to controlling pressure.

III. Applications for Microfluidic Free Interface Diffusion

Microfluidic free interfaces created in accordance with embodiments ofthe present invention may be useful in a variety of applications. Asspecifically described in detail below, the exclusively diffusive mixingthat occurs at such a microfluidic free interface may provide conditionsadvantageous in the formation of crystals, as well as in the performanceof certain assays.

In general, microfluidics enables the handling of fluids on thesub-nanoliter scale. Consequently, there is no need to use largecontainment chambers, and hence, assays may be performed on thenanoliter, or subnanoliter scale. The utilization of extremely smallvolumes allows for thousands of assays to be performed while consumingthe same sample volume required for one macroscopic free-interfacediffusion experiment. This reduces costly and time-consumingamplification and purification steps, and makes possible the screeningof proteins that are not easily expressed, and hence must be purifiedfrom a bulk sample.

Microfluidics further offers savings in preparation times, as hundreds,or even thousands of assays may be performed simultaneously. The use ofscaleable metering techniques as previously described, allow forparallel experimentation to be conducted without increased complexity incontrol mechanisms.

Finally, other researchers have proposed the diffusion of materialsacross an interface between two flowing liquids. Kamholz et al.,“Quantitative Analysis of Molecular Interaction in a MicrofluidicChannel”, Anal. Chem. Vol. 71, No. 20 (1999). However, such an approachrequires relatively large volumes of sample owing to the continuous flowof materials, and also involves the exposure of large volumes of theflowing liquid to the relatively steep concentration gradient present atthe fluidic interface. Embodiments in accordance with the presentinvention utilizing diffusion across a microfluidic free interface avoidthese and other problems associated with diffusion between flowingmaterials.

1. Protein Crystallography

The use of microfluidic free interface diffusion techniques in proteincrystallography has previously been discussed in detail in U.S.nonprovisional patent application Ser. No. 09/887,997 filed Jun. 22,2001; and U.S. nonprovisional patent application Ser. No. 10/117,978filed Apr. 5, 2002. These applications are hereby incorporated byreference for all purposes.

As employed in the following discussion, the term “crystallizing agent”describes a substance that is introduced to a solution of targetmaterial to lessen solubility of the target material and thereby inducecrystal formation. Crystallizing agents typically includecountersolvents in which the target exhibits reduced solubility, but mayalso describe materials affecting solution pH or materials such aspolyethylene glycol that effectively reduce the volume of solventavailable to the target material. The term “countersolvent” is usedinterchangeably with “crystallizing agent”.

Briefly, crystallization is an important technique to the biological andchemical arts. A high-quality crystal of a target compound can beanalyzed by x-ray diffraction techniques to produce an accuratethree-dimensional structure of the target. This three-dimensionalstructure information can then be utilized to predict functionality andbehavior of the target.

In theory, the crystallization process is simple. A target compound inpure form is dissolved in or exchanged into some type of solvent. Forproteins, this solvent is typically, although not always, water. Forsmall molecule chemicals, the solvent can be water- or organic-based.The chemical environment of the dissolved target material is thenaltered such that the target is less soluble and reverts to the solidphase in crystalline form. This change in chemical environment typicallyaccomplished by introducing a crystallizing agent that makes the targetmaterial is less soluble, although changes in temperature and pressurecan also influence solubility of the target material.

In practice however, forming a high quality crystal is generallydifficult and sometimes impossible, requiring much trial and error andpatience on the part of the researcher. Specifically, the highly complexstructure of even simple biological compounds means that they are notamenable to forming a highly ordered crystalline structure. Therefore, aresearcher must be patient and methodical, experimenting with a largenumber of conditions for crystallization, altering parameters such assample concentration, solvent type, countersolvent type, temperature,and duration in order to obtain a high quality crystal, if in fact acrystal can be obtained at all.

Systems and methods in accordance with embodiments of the presentinvention are particularly suited to crystallizing larger biologicalmacromolecules or complexes thereof, such as proteins, nucleic acids,viruses, and protein/ligand assemblies. However, crystallization inaccordance with the present invention is not limited to any particulartype of target material.

In accordance with embodiments of the present invention, acrystallization technique called gated micro free interface diffusion(Gated μ-FID), has been developed. Referring back to FIGS. 34A-D, onegeneric embodiment of protein crystallization utilizing microfluidicfree interface diffusion introduces a sample solution 9606 into firstflow channel portion 9600 a under pressure, and introduces a solutioncontaining a countersolvent or crystallizing agent into second flowchannel portion 9600 b under pressure. Because of the gas permeabilityof the surrounding elastomer material 9607, gas 9604 is displaced by theincoming solutions 9608 and 9610 and out-gasses through elastomer 9607.

As shown in FIG. 34C, the pressurized out-gas priming of flow channelportions 9600 a and 9600 b allows uniform filling of these dead-endedflow channel portions without air bubbles. Upon deactuation of valve9602 as shown in FIG. 34D, a microfluidic free interface 9612 isdefined, thereby allowing for formation of a diffusion gradient betweenthe sample solution and the solution containing the crystallizing agent.As a result of diffusive mixing between the sample and the crystallizingagent, the solution environment is gradually changed, resulting in theformation of protein crystals in the channel.

The formation of protein crystals utilizing gated μ-FID retains theefficient sampling of phase space achieved by macroscopic free interfacediffusion techniques, with a number of added advantages, including theparsimonious use of sample solutions, ease of set-up, creation of welldefined fluidic interfaces, control over equilibration dynamics, and theability to conduct high-throughput parallel experimentation.

Another possible advantage of the formation of protein crystalsutilizing gated μ-FID is the formation of high quality crystals, asillustrated in connection with FIGS. 44A and 44B. FIG. 44A shows asimplified schematic view of a protein crystal being formed utilizing aconventional macroscopic free interface diffusion technique.Specifically, nascent protein crystal 9200 is exposed to sample fromsolution 9202 that is experiencing a net conductive flow of sample as aresult of the action of buoyant forces. As a result of thedirectionality of this conductive flow, the growth of protein crystal9200 is also directional. However, as described in Nerad et al.,“Ground-Based Experiments on the Minimization of Convection During theGrowth of Crystals from Solution”, Journal of Crystal Growth 75, 591-608(1986), assymetrical growth of a protein crystal can give rise tounwanted strain in the lattice of the growing crystal, promotingdislocations and/or the incorporation of impurities within the lattice,and otherwise adversely affecting the crystal quality.

By contrast, FIG. 44B shows a simplified schematic view of a proteincrystal being formed utilizing diffusion across a microfluidic freeinterface in accordance with an embodiment of the present invention.Nascent protein crystal 9204 is exposed to sample solution 9206 that isdiffusing within the crystallizing agent. This diffusion isnondirectional, and the growth of protein crystal 9204 is alsocorrespondingly nondirectional. Accordingly, the growing crystal avoidsstrain on the lattice and the attendant incorporation of impurities anddislocations experienced by the growing crystal shown in FIG. 44A.Accordingly, the quality of the crystal in FIG. 44B is of high quality.

Typical targets for crystallization are diverse. A target forcrystallization may include but is not limited to: 1) biologicalmacromolecules (cytosolic proteins, extracellular proteins, membraneproteins, DNA, RNA, and complex combinations thereof), 2) pre- andpost-translationally modified biological molecules (including but notlimited to, phosphorylated, sulfolated, glycosylated, ubiquitinated,etc. proteins, as well as halogenated, abasic, alkylated, etc. nucleicacids); 3) deliberately derivatized macromolecules, such as heavy-atomlabeled DNAs, RNAs, and proteins (and complexes thereof),selenomethionine-labeled proteins and nucleic acids (and complexesthereof), halogenated DNAs, RNAs, and proteins (and complexes thereof),4) whole viruses or large cellular particles (such as the ribosome,replisome, spliceosome, tubulin filaments, actin filaments, chromosomes,etc.), 5) small-molecule compounds such as drugs, lead compounds,ligands, salts, and organic or metallo-organic compounds, and 6)small-molecule/biological macromolecule complexes (e.g., drug/proteincomplexes, enzyme/substrate complexes, enzyme/product complexes,enzyme/regulator complexes, enzyme/inhibitor complexes, and combinationsthereof). Such targets are the focus of study for a wide range ofscientific disciplines encompassing biology, biochemistry, materialsciences, pharmaceutics, chemistry, and physics.

During crystallization screening, a large number of chemical compoundsmay be employed. These compounds include salts, small and largemolecular weight organic compounds, buffers, ligands, small-moleculeagents, detergents, peptides, crosslinking agents, and derivatizingagents. Together, these chemicals can be used to vary the ionicstrength, pH, solute concentration, and target concentration in thedrop, and can even be used to modify the target. The desiredconcentration of these chemicals to achieve crystallization is variable,and can range from nanomolar to molar concentrations. A typicalcrystallization mix contains set of fixed, but empirically-determined,types and concentrations of ‘precipitants’, buffers, salts, and otherchemical additives (e.g., metal ions, salts, small molecular chemicaladditives, cryo protectants, etc.). Water is a key solvent in manycrystallization trials of biological targets, as many of these moleculesmay require hydration to stay active and folded.

‘Precipitating’ agents act to push targets from a soluble to insolublestate, and may work by volume exclusion, changing the dielectricconstant of the solvent, charge shielding, and molecular crowding.Precipitating agents compatible with the PDMS material of certainembodiments of the chip include, but are not limited to, salts, highmolecular weight polymers, polar solvents, aqueous solutions, highmolecular weight alcohols, and divalent metals.

Precipitating compounds, which include large and small molecular weightorganics, as well as certain salts are used from under 1% to upwards of40% concentration, or from less than 0.5M to greater than 4Mconcentration. Water itself can act in a precipitating manner forsamples that require a certain level of ionic strength to stay soluble.Many precipitants may also be mixed with one another to increase thechemical diversity of the crystallization screen. The microfluidicsdevices described in this document are readily compatible with a broadrange of such compounds. Moreover, many precipitating agents (such aslong- and short-chain organics) are quite viscous at highconcentrations, presenting a problem for most fluid handling devices,such as pipettes or robotic systems. The pump and valve action ofmicrofluidics devices in accordance with embodiments of the presentinvention enable handling of viscous agents.

An investigation of solvent/precipitating agent compatibility withparticular elastomer materials may be conducted to identify optimumcrystallizing agents, which may be employed develop crystallizationscreening reactions tailored for the chip that are more effective thanstandard screens.

Solution pH can be varied by the inclusion of buffering agents; typicalpH ranges for biological materials lie anywhere between values of3.5-10.5 and the concentration of buffer, generally lies between 0.01and 0.25 M. The microfluidics devices described in this document arereadily compatible with a broad range of pH values, particularly thosesuited to biological targets.

A nonexclusive list of possible buffers is as follows: Na/K-Acetate;HEPES; Na-Cacodylate; Na/K-Citrate; Na/K-Succinate; Na/K-Phosphate;TRIS; TRIS-Maleate; Imidazole-Maleate; BisTrisPropane; CAPSO, CHAPS,MES, and imidizole.

Additives are small molecules that affect the solubility and/or activitybehavior of the target. Such compounds can speed crystallizationscreening or produce alternate crystal forms of the target. Additivescan take nearly any conceivable form of chemical, but are typically monoand polyvalent salts (inorganic or organic), enzyme ligands (substrates,products, allosteric effectors), chemical crosslinking agents,detergents and/or lipids, heavy metals, organo-metallic compounds, traceamounts of precipitating agents, and small and large molecular weightorganics.

Many of these chemicals can be obtained in predefined screening kitsfrom a variety of vendors, including but not limited to Hampton Researchof Laguna Niguel, Calif., Emerald Biostructures of Bainbridge Island,Wash., and Jena BioScience of Jena, Germany, that allow the researcherto perform both ‘sparse matrix’ and ‘grid’ screening experiments. Sparsematrix screens attempt to randomly sample as much of precipitant,buffer, and additive chemical space as possible with as few conditionsas possible. Grid screens typically consist of systematic variations oftwo or three parameters against one another (e.g., precipitantconcentration vs. pH). Both types of screens have been employed withsuccess in crystallization trials, and the majority of chemicals andchemical combinations used in these screens are compatible with the chipdesign and matrices in accordance with embodiments of the presentinvention.

Moreover, current and future designs of microfluidic devices may enableflexibly combinatorial screening of an array of different chemicalsagainst a particular target or set of targets, a process that isdifficult with either robotic or hand screening. This latter aspect isparticularly important for optimizing initial successes generated byfirst-pass screens.

In addition to chemical variability, a host of other parameters can bevaried during crystallization screening. Such parameters include but arenot limited to: 1) volume of crystallization trial, 2) ratio of targetsolution to crystallization solution, 3) target concentration, 4)cocrystallization of the target with a secondary small or macromolecule,5) hydration, 6) incubation time, 7) temperature, 8) pressure, 9)contact surfaces, 10) modifications to target molecules, and 11)gravity.

Volumes of crystallization trials can be of any conceivable value, fromthe picoliter to milliliter range. Typical values may include but arenot limited to: 0.1, 0.2, 0.25, 0.4, 0.5, 0.75, 1, 2, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200,225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 700, 750, 800, 900,1000, 1100, 1200, 1250, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2250, 2500, 3000, 4000, 5000, 6000, 7000, 7500, 8000, 9000, and 10000nL. The microfluidics devices previously described can access thesevalues.

In particular, access to the low volume range for crystallization trials(<100 nL) is a distinct advantage of embodiments of the microfluidicschips in accordance with embodiments of the present invention, as suchsmall-volume crystallization chambers can be readily designed andfabricated, minimizing the need the need for large quantities ofprecious target molecules. The low consumption of target material ofembodiments in accordance with the present invention is particularlyuseful in attempting to crystallize scarce biological samples such asmembrane proteins, protein/protein and protein/nucleic acid complexes,and small-molecule drug screening of lead libraries for binding totargets of interest.

The ratios of a target solution to crystallization mix can alsoconstitute an important variable in crystallization screening andoptimization. These rations can be of any conceivable value, but aretypically in the range of 1:100 to 100:1target:crystallization-solution. Typical target:crystallization-solutionor crystallization-solution:target ratios may include but are notlimited to: 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:25, 1:20,1:15, 1:10, 1:9, 1:8, 1:7.5, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2.5, 1:2, 1:1,2:3, 3:4, 3:5, 4:5, 5:6, 5:7, 5:9, 6:7, 7:8, 8:9, and 9:10. Aspreviously described, microfluidics devices in accordance withembodiments of the present invention can be designed to access multipleratios simultaneously on a single chip.

Target concentration, like crystallization chemical concentration, canlie in a range of values and is an important variable in crystallizationscreening. Typical ranges of concentrations can be anywhere from <0.5mg/ml to >100 mg/ml, with most commonly used values between 5-30 mg/ml.The microfluidics devices in accordance with embodiments of the presentinvention are readily compatible with this range of values.

Cocrystallization generally describes the crystallization of a targetwith a secondary factor that is a natural or nonnatural binding partner.Such secondary factors can be small, on the order of about 10-1000 Da,or may be large macromolecules. Cocrystallization molecules can includebut are not limited to small-molecule enzyme ligands (substrates,products, allosteric effectors, etc.), small-molecule drug leads,single-stranded or double-stranded DNAs or RNAs, complement proteins(such as a partner or target protein or subunit), monoclonal antibodies,and fusion-proteins (e.g., maltose binding proteins, glutathioneS-transferase, protein-G, or other tags that can aid expression,solubility, and target behavior). As many of these compounds are eitherbiological or of a reasonable molecular weight, cocrystallizationmolecules can be routinely included with screens in the microfluidicschips. Indeed, because many of these reagents are expensive and/or oflimited quantity, the small-volumes afforded by the microfluidics chipsin accordance with embodiment of the present invention make them ideallysuited for cocrystallization screening.

Hydration of targets can be an important consideration. In particular,water is by far the dominant solvent for biological targets and samples.The microfluidics devices described in this document are relativelyhydrophobic, and are compatible with water-based solutions.

The length of time for crystallization experiments can range fromminutes or hours to weeks or months. Most experiments on biologicalsystems typically show results within 24 hours to 2 weeks. This regimeof incubation time can be accommodated by the microfluidics devices inaccordance with embodiments of the present invention.

The temperature of a crystallization experiment can have a great impacton success or failure rates. This is particularly true for biologicalsamples, where temperatures of crystallization experiments can rangefrom 0-42° C. Some of the most common crystallization temperatures are:0, 1, 2, 4, 5, 8, 10, 12, 15, 18, 20, 22, 25, 30, 35, 37, and 42.Microfluidics devices in accordance with embodiments of the presentinvention can be stored at the temperatures listed, or alternatively maybe placed into thermal contact with small temperature control structuressuch as resistive heaters or Peltier cooling structures.

In addition, the small footprint and rapid setup time of embodiments inaccordance with the present invention allow faster equilibration todesired target temperatures and storage in smaller incubators at a rangeof temperatures. Moreover, as the microfluidics systems in accordancewith embodiments of the present invention do not place thecrystallization experiment in contact with the vapor phase, condensationof water from the vapor phase into the drop as temperatures change, aproblem associated with conventional macroscopic vapor-diffusiontechniques, is avoided. This feature represents an advance over manyconventional manual or robotic systems, where either the system must bemaintained at the desired temperature, or the experiment must remain atroom temperature for a period before being transferred to a newtemperature.

Variation in pressure is an as yet understudied crystallizationparameter, in part because conventional vapor-diffusion and microbatchprotocols do not readily allow for screening at anything typically otherthan atmospheric pressure. The rigidity of the PDMS matrix enablesexperiments to probe the effects of pressure on target crystallizationon-chip.

The surface on which the crystallization ‘drop’ sits can affectexperimental success and crystal quality. Examples of solid supportcontact surfaces used in vapor diffusion and microbatch protocolsinclude either polystyrene or silanized glass. Both types of supportscan show different propensities to promote or inhibit crystal growth,depending on the target. In addition, the crystallization ‘drop’ is incontact with either air or some type of poly-carbon oil, depending onwhether the experiment is a vapor-diffusion or microbatch setup,respectively. Air contact has the disadvantage in that free oxygenreacts readily with biological targets, which can lead to proteindenaturation and inhibit or degrade crystallization success. Oil allowstrace hydrocarbons to leach into the crystallization experiment, and cansimilarly inhibit or degrade crystallization success.

Microfluidics device designs in accordance with embodiments of thepresent invention may overcome these limitations by providing anonreactive, biocompatible environment that completely surrounds thecrystallization reaction. Moreover, the composition of thecrystallization chambers in the microfluidics chips can conceivably bevaried to provide new surfaces for contacting the crystallizationreaction; this would allow for routine screening of different surfacesand surface properties to promote crystallization.

Crystallization targets, particularly those of biological origin, mayoften be modified to enable crystallization. Such modifications includebut are not limited to truncations, limited proteolytic digests,site-directed mutants, inhibited or activated states, chemicalmodification or derivatization, etc. Target modifications can be timeconsuming and costly; modified targets require the same thoroughscreening as do unmodified targets. Microfluidics devices of the presentinvention work with such modified targets as readily as with theoriginal target, and provide the same benefits.

The effect of gravity as a parameter for crystallization is yet anotherunderstudied crystallization parameter, because of the difficulty ofvarying such a physical property. Nonetheless, crystallizationexperiments of biological samples in zero gravity environments haveresulted in the growth of crystals of superior quality than thoseobtained on Earth under the influence of gravity.

The absence of gravity presents problems for traditional vapor-diffusionand microbatch setups, because all fluids must be held in place bysurface tension. The need to often set up such experiments by hand alsoposes difficulties because of the expense of maintaining personnel inspace. Microfluidics devices in accordance with embodiments of thepresent invention, however, would enable further exploration ofmicrogravity as a crystallization condition. A compact, automatedmetering and crystal growth system would allow for: 1) launching ofsatellite factory containing target molecules in a cooled, but liquidstate, 2) distribution of targets and growth of crystals, 3) harvestingand cryofreezing of resultant crystals, and 4) return of cryo-storedcrystals to land-based stations for analysis.

Embodiments of crystallization structures and methods in accordance withthe present invention offer a number of advantages over conventionalapproaches. One advantage is that the extremely small volumes(nanoliter/sub-nanoliter) of sample and crystallizing agent permit awide variety of recrystallization conditions to be employed utilizing arelatively small amount of sample.

Another advantage of crystallization structures and methods inaccordance with embodiments of the present invention is that the smallsize of the crystallization chambers allows crystallization attemptsunder hundreds or even thousands of different sets of conditions to beperformed simultaneously. The small volumes of sample and crystallizingagent employed in crystallization also result in a minimum waste ofvaluable purified target material.

A further advantage of crystallization in accordance with embodiments ofthe present invention is relative simplicity of operation. Specifically,control over flow utilizing parallel actuation requires the presence ofonly a few control lines, with the introduction of sample andcrystallizing agent automatically performed by operation of themicrofabricated device permits very rapid preparation times for a largenumber of samples.

Still another advantage of crystallization systems in accordance withembodiments of the present invention is the ability to control solutionequilibration rates. Crystal growth is often very slow, and no crystalswill be formed if the solution rapidly passes through an optimalconcentration on the way to equilibrium. It may therefore beadvantageous to control the rate of equilibration and thereby promotecrystal growth at intermediate concentrations. In conventionalapproaches to crystallization, slow-paced equilibrium is achieved usingsuch techniques as vapor diffusion, slow dialysis, and very smallphysical interfaces.

Another potential advantage of the creation and use of microfluidic freeinterfaces in accordance with embodiments of the present invention isthe ability to preserve intact crystals that have formed as a result ofthe diffusive mixing. Specifically, once a crystal has nucleated andgrown, it is frequently desirable to reduce the temperature of thecrystal in order to preserve its structure during subsequent x-rayanalysis. Such analysis may induce radiation damage, which undesirablydecays or destroys the crystal.

Accordingly, it is desirable to expose the crystal to a cryogen or otherlow temperature compound to preserve the crystal intact. The exclusivelydiffusive mixing allowed by embodiments of microfluidic free interfacesin accordance with embodiments of the present invention allow for theexposure of such cryogens to the crystal, such that the reduction intemperature does not result in damage or change in the crystalstructure. For example, in certain embodiments in accordance with thepresent invention, once the presence of a crystal has been identified, asecond microfluidic free interface between the crystal-containingsolution and a cryogenic solution can be created. The cryogenic contentsof that chamber can then be allowed to diffuse into the crystallizationchamber. In this scheme, large number of cryogenic types andconcentrations can readily be screened without changing the location ofthe crystal in-situ.

2. Solubility Studies (Proteomics)

The above discussion has focused upon protein crystallizationapplications wherein the environment of the solvent containing a proteinsample was altered by diffusion of a crystallizing agent across amicrofluidic free interface, resulting in a change in phase of theprotein and the formation of a crystal. However, an important precursorto such a crystallization study is identifying and obtaining a solutionin which the protein is sufficiently soluble and mono-disperse to form acrystal upon exposure to a crystallizing agent.

Accordingly, an alternative application for microfluidic free interfacediffusion in accordance with an embodiment of the present invention isto determine solubility of a protein in various solvents. Specifically,a free microfluidic interface could be utilized to meter the flow of asolvent to a protein sample, and thus to identify the solubilitycharacteristic of the protein. Specifically, the presence of a freemicrofluidic interface would create a uniform and precise concentrationgradient along a channel of known volume, thereby enabling a researcherto identify with precision the ability of a known amount of proteinsample entering into solution. This information could in turn beutilized to allow for the creation of concentrated protein solutionsthat are in turn susceptible to forming crystals.

3. Sample Purification Through Quasi-Dialysis

The above discussion has focused upon protein crystallization andproteomics solubility applications wherein the relative amount of sampleand solvent are precisely controlled to govern concentration of thesample in solution, and hence the propensity of the sample to emergefrom solution in solid, crystalline form. However, a number of factorsother than sample concentration may also play an important role insample behavior.

One such factor is sample purity. One artifact of many common processesfor protein isolation and/or purification is the concentration of smallsalts with the sample. These salts can stabilize a protein in solution,and otherwise interfere with crystal nucleation and growth. Oneconventional approach for removing these salts is through dialysisacross a membrane. However, this approach typically takes place on themacroscopic scale, involving the use of relatively large sample volumesand involving the coarse removal of bulk quantities of salts.

Thus yet another potential alternative application for microfluidic freeinterface diffusion in accordance with an embodiment of the presentinvention is in the removal of unwanted components from a samplesolution. For example, a solution comprising isolated and/or purifiedprotein that contains a significant concentration of small salts may bepositioned in a microfluidic channel on one side of a valve. Upondeactuation of the valve, a microfluidic free interface between theprotein solution and a diluent may be created, such that the smallersalts rapidly diffuse into the diluent and are thereby depleted in thesample. Of course, some amount of protein present in the sample wouldalso be expected to diffuse across the microfluidic free interface andthereby be lost to the diluent during this process. However, disparityin size between the small salt and the large protein molecule would beexpected to constrain relative diffusion rates to limit the loss ofprotein in the sample.

4. Growth of Other than Protein Crystals

Embodiments in accordance with the present invention have focused thusfar on the growth of crystals biological macromolecules such asproteins. However, other types of molecules also form crystals whosethree dimensional structure may be studied. One such promising candidatefor crystal formation are inorganic nanocrystals which may be utilizedto entrap single atoms.

Recently the scientific community has exhibited interest in growingnano-crystals from inorganic materials. Such crystals have dimensions onthe nanometer scale, and in some instances are able to encloseindividual atoms. These crystals exhibit optical properties such asabsorption and emission of light, which are governed by quantummechanical effects. These optical properties may be controlled or variedaccording to such factors as the size and quality of the crystal, andthe identity of the atomic species enclosed thereby.

As with a protein crystal, the size and quality of such a nano-crystaldepends to a large degree upon conditions in which it is grown.Therefore, it may prove important to control both the phase-spaceevolution, and the rate of this evolution during growth of anano-crystal. Such control can be accomplished utilizing microfluidicfree interface diffusion in accordance with embodiments of the presentinvention, as has been described previously in connection with proteincrystallization.

Moreover, in certain cases it may be prove of interest to determine theeffect upon crystal properties of changing the concentration of oneelement of the crystallization. This may be accomplished usingmicrofluidic free interface diffusion in accordance with embodiments ofthe present invention by setting up a gradient through a string ofconnected chambers, with a large reservoir at either end (or simplyalong a channel connecting two large reservoirs). Crystals are thengrown at each condition (or along a continuum of conditions) and thenare interrogated optically to determine their absorption/emissionproperties. In this manner, it is possible to independently vary asingle crystallization parameter (such as the concentration of a sample,a crystallizing agent, a precipitant, or some other species).

It is also possible to grow on chip a spatial distribution ofnanocrystals with different optical properties.

5. Diffusive Immunoassays

Another potentially valuable application for microfluidic freeinterfaces in accordance with embodiments of the present invention isanalysis of binding of analytes to targets. Conventionally, binding ofan analyte to a target has been determined through techniques such aschromatography, wherein the changed velocity of an analyte/targetcombination relative to either of the analyte or target alone in aconvective flow is employed to identify the presence of the boundcombination.

Embodiments in accordance with the present invention exploit the factthat the binding of analyte to a target will also result in a changedsize and hence coefficient of diffusion of the analyte/targetcombination relative to either of the analyte or target alone. Thischanged diffusion coefficient can be utilized to identify the boundcombination.

FIG. 45A shows a simplified plan view of one embodiment of a device thatis able to detect diffusion across a microfluidic free interface inaccordance with the present invention. Chambers 8000 and 8002 containingfluids A and B respectively are connected by channel 8004 having a widththat is restricted in at least one dimension. Valve 8006, which isinitially closed, is positioned within channel 8004 to maintain fluids Aand B separate. Chamber 8003 is in fluid communication with chamber 8002through a connecting channel 8005 and also contains fluid B.

First fluid A contains an analyte 8008 detectable through analyticaltechniques, including but not limited to detecting changes in fluoresce,refractive index, conductivity, light-scattering, and the use ofcolorimetric sensors. Second fluid B contains a target 8010 that isknown to bind to analyte 8008. Upon binding of analyte 8008 to target8010, the analyte/target combination exhibit a substantially reducedcoefficient of diffusion relative to that of the analyte alone.

At an initial time T₀, valve 8006 is opened to create a microfluidicfree interface 8012 between fluids A and B. Commencing at time T₁,analyte 8008 begins to diffuse through channel 8004, chamber 8002, andchannel 8005 into chamber 8003. Absent a binding reaction between theanalyte 8008 and target 8010, the analyte would be expected to beginappearing in end chamber 8003 at a given rate over time. This is shownin FIG. 45B, which plots intensity of a signal expected to be detectedin the end chamber 8003 versus time of diffusion.

However, because some reaction occurs in the channel between analyte8008 and target 8010, some percentage of analyte 8008 binds to target8010, effectively slowing the rate of diffusion of this percentage ofthe analyte. As a result, the observed intensity of the signal in theend chamber over time will be reduced, reflecting the slower rate ofdiffusion of the bound analyte/target combination. This is also shown inFIG. 45B, which plots intensity of an observed signal in the secondchamber versus time of diffusion.

The difference between expected and observed signal intensity over timemay be analyzed to reveal potentially valuable information regarding thefluidic system. For example, where the concentration of target andanalyte in the respective solutions is known, analysis of evolution overtime of the intensity signal can indicate the reactivity between thetarget and analyte, i.e. the rate at which the analyte binds to thetarget and whose diffusion is slowed as result. Alternatively, wherereactivity between the target and the analyte is known, analysis of thetime evolution of the intensity signal can indicate the initialconcentration of the target. Further alternatively, where initialconcentrations and reactivity are known, analysis of time evolution ofthe intensity signal can indicate the size of the target, as manifestedby a reduced diffusion rate of the bound analyte/target combinationversus the analyte alone.

The example just provided utilized an analysis of the time evolution ofintensity of a signal from a diffusing analyte. However, embodiments inaccordance with the present invention are not limited to this particularapproach.

For example, FIG. 46 shows an alternative embodiment of a structure foruse in diffusive assays. Structure 8100 comprises microfluidic channel8102 of constant width and depth that is bisected by valve 8104. Fluid Acontaining detectable analyte 8106 is positioned on one side of valve8104. Fluid B containing target 8108 is positioned on the other side ofvalve 8104.

At an initial time T₀, valve 8104 opened to create a microfluidic freeinterface 8110 between fluids A and B. Commencing at time T₁, analyte8106 begins to diffuse across interface 8110. Absent a binding reactionbetween the analyte 8106 and target-8108, after a certain time theprofile of intensity of the signal over the distance of the channelwould be expected to exhibit a characteristic shape. This is shown inFIG. 46B, which plots intensity of a signal expected to be detected inthe second chamber, versus distance of diffusion.

However, because some reaction occurs between analyte 8106 and target8108, some percentage of the analyte binds to the target, effectivelyslowing the rate of diffusion of this percentage of the analyte. As aresult, the observed intensity of the signal in the second half of thechannel would be changed, reflecting the slower rate of diffusion of thebound analyte/target combination. This is also shown in FIG. 46B, whichplots intensity of an observed signal in the second chamber versusdistance of diffusion.

As with the embodiment shown in FIGS. 45A-B, the difference betweenexpected and observed signal intensity over distance may be analyzed toreveal potentially valuable information. For example, where theconcentration of target and analyte in the respective solutions isknown, analysis of spatial evolution of the intensity signal canindicate the reactivity between the target and analyte, i.e. the rate atwhich the analyte binds to the target and whose diffusion is slowed asresult. Alternatively, where reactivity between the target and theanalyte is known, analysis of spatial evolution of the intensity signalcan indicate the initial concentration of the target. Furtheralternatively, where initial concentrations and reactivity are know,analysis of spatial evolution of the intensity signal can indicate themass of the target, as manifested by reduction in the rate of diffusionof the bound analyte/target combination versus the analyte alone.

6. Viscosity Measurement

The above discussion focused upon identifying properties of a chemicalsample such as concentration or reactivity based upon diffusion of thesample across a microfluidic free interface under known conditions. Inaccordance with alternative embodiments of the present inventionhowever, properties of the fluid in which diffusion is taking place,rather than of the sample diffusing within the fluid, may be determinedby observing the character of diffusion across a free microfluidicinterface.

For example, one conventional approach to measuring viscosity calls forinserting a piston into a closed vessel containing the sample fluid, andthen measuring the torque required to rotate the piston in the vessel.While this approach is adequate for measuring the viscosity of largerfluid samples, it offers the disadvantage of requiring and consumingrelatively large volumes of sample fluid. Such volumes may not beavailable for analysis, particularly in the context of biologicalanalysis.

In accordance with an alternative embodiment of the present invention,diffusion of a marker of known size and diffusion coefficient (i.e. afluorescently labeled bead or macromolecule) across a microfluidic freeinterface created within the fluid may be utilized to determine itsviscosity. Such a technique is particularly applicable to analysis ofthe viscosity of biological or physiological samples, as the dimensionsof the microfluidic channels in which diffusion occurs occupiesrelatively small volumes.

7. Competitive Binding Assay

A different type of assay from the diffusive immunoassay discussed aboveis the competitive binding assay. A competitive binding assay determinesthe propensity of a competitor ligand to displace an existing ligandbound to a target molecule such as a protein. This displacement tendencymay be determined by mixing the original ligand/target moleculecombination with the competitor ligand at various concentrations.Conventionally, such binding assays require a large number of separateexperiments to cover a suitable range of concentrations at sufficientresolution.

However, diffusion across a microfluidic free interface in accordancewith an embodiment of the present invention allows for establishingcontinuous or discrete gradients such that a large number of assayconditions can be sampled at once. For example, in accordance with oneembodiment of the present invention, a competitive binding assay may beperformed utilizing a discrete gradient of competitor ligandconcentrations created in a string of chambers connected by channels.

This approach is illustrated in FIG. 49, which shows a string ofchambers 8300 a-c connected by narrow channels 8302 and positionedbetween reservoirs 8304 and 8306. Reservoir 8304 contains a highconcentration the competitor ligand 8308, and reservoir 8306 does notcontain the competitor ligand. Flow into each of chambers 8300 a-c fromthe reservoirs or from each other may be independently controlled byvalves 8305 located in connecting channels 8302. Concentrations ateither end of the string of chambers may be fixed by the large volumesof the reservoirs relative to the chambers, or by continuous flows ofmaterial to the reservoirs.

Initially, the chambers in the string are charged with a first fluidcontaining a certain concentration of the original ligand 8310 bound totarget 8312. Next, chambers 8300 a-c are placed in fluid communicationwith the reservoirs and with each other by opening interface valves8305. Once these interface valves are opened, a microfluidic freeinterface is established between the chambers, and the solutionsequilibrate by diffusion. The position of each chamber along the stringdetermines the concentration of the competitor ligand in that particularchamber.

The competitor ligand is typically much smaller than the target moleculeand therefore equilibrates quickly. The original ligand displaced fromthe target by the competitive ligand is free to diffuse from the targetchamber to another chamber. Owing to reduced size of the original ligandrelative to the ligand/target combination, the displaced original ligandwould be expected to exhibit a greater diffusion coefficient and hencediffuse at a faster rate. In a manner similar to that described above inconnection with FIGS. 45A-B and 46A-B, if the original ligand isfluorescent, its presence and hence the rate of displacement from thetarget may be revealed by analyzing fluorescence signals for deviationfrom expected temporal or spatial profiles.

While the embodiment just described monitors changes in location of afluorescent signal over time to identify competitive binding, inaccordance with alternative embodiments of the present invention, adiminuation in intensity of a fluorescent signal may also be detected toperform competitive binding assays. Specifically, fluorescent ligandsmay be utilized in the presence of quenching molecules. A fluorescentmolecule is quenched when it interacts another molecule to suppressfluorescent emission.

FIG. 50 shows a plan view of a simplified embodiment of a microfluidicsystem for performing competitive binding assays utilizing a quenchingmaterial. First chamber 8400 contains target molecule 8402, firstfluorescent ligand 8404, and quenching molecule 8406. First chamber 8400is connected via a microfabricated channel 8408 to a second chamber 8410containing competitor ligand 8412, and the same concentrations of targetmolecule 8402, first fluorescent ligand 8404, and quenching molecule8406 as are present in first chamber 8400.

When first fluorescent ligand 8404 is bound to target 8402, it cannotinteract with the quenching molecule and thus a fluorescent emission maybe observed. When the first fluorescent ligand is not bound to thetarget, however, it is quenched and therefore no fluorescent emission isobserved. In this manner, a decline in fluorescent signal intensity mayindicate competitive binding behavior.

Valve 8414 originally separating the contents of chambers 8400 and 8410is opened, creating microfluidic free interface 8414 and allowing thetwo fluids to mix by diffusion. Since the fluids are identical exceptfor the presence of the competitor ligand in the second chamber, onlythe competitor ligand experiences net diffusive transport between thechambers and establishes a linear gradient between the chambers. Byobserving the fluorescent intensity along different portions of thechannel, the concentration of competitor ligand necessary to displacethe first fluorescent ligand can be determined. Alternatively, the levelof fluorescence in the end chambers over time can be monitored todetermine concentration of the competitor ligand necessary to displacethe first fluorescent ligand from the target. Using either method, asingle experiment may be employed to determine the outcome of thecompetitive binding assay under a continuum of conditions.

8. Substrate/Inhibitor Assays

Activity of an enzyme can be measured in response to the presence ofdifferent concentrations of a ligand. For example, a protein catalyzinga reaction may be impaired by a small molecule that binds strongly tothe active site of the protein. A substrate turnover assay may be usedto determine the effect of such a ligand on enzyme activity.

Where the enzyme catalyses a reaction that creates a fluorescentproduct, the activity of the enzyme may be determined by monitoringfluorescence. One example of catalysis of a fluorescence-producingreaction is the hydrolyzation of Beta-D-galactopyranoside into resorufinby the enzyme Beta-galactosidase. Resorufin is a fluorescent productthat may be monitored over time. Ramsey et. al, Analytical Chem. 69,3407-3412 (1997). By determining activity of the enzyme as a function ofthe concentration of an inhibiting molecule, the Michaelis-Menten rateconstants of the reaction can be determined. The generation of adiffusion gradient across a microfluidic free interface in accordancewith an embodiment of the present invention can enable determination ofthis concentration dependence in a single experiment.

FIG. 51 shows a simplified plan view of an embodiment of a microfluidicstructure for performing such a substrate turnover assay. Again,reservoirs 8500 and 8502 are connected by channel 8504 having a widththat is much smaller than the dimensions of the chambers. Reservoirs8500 and 8502 contain substrate 8509 at the same concentration, andreservoir 8502 also contains inhibitor molecule 8508. A gradient ofinhibitor molecule concentration is achieved by maintaining a fixedconcentration of material in the reservoirs 8500 and 8502, due either totheir large volumes relative to the channel, or to replenishment ofmaterial from an external source. Chambers may or may not be includedalong the length of channel 8504 to provide larger volumes for reactionsites at particular concentrations.

Once the concentration gradient of inhibitor molecule has beenestablished, valves 8510 are actuated to isolate sections of channel (orindividual chambers connected by the channel) that are subsequentlymixed with separate wells 8512 containing enzyme 8514. By monitoringfluorescence of the each well over time, it is possible to determine theactivity the enzyme in each chamber.

Detection of activity in accordance with embodiments of the presentinvention is not required to be through fluorescence. Depending upon thesubstrate to be identified, enzyme activity and the resulting catalysiscould be determined through analysis of colorimetry, index ofrefraction, surface plasmon resonance, or conductivity, to name only afew.

9. Bioreactor

Only a small percentage of known microorganisms can be cultured andgrown in a laboratory setting. This relatively low success rate islargely attributable to the substantial number of factors typicallyrequired to induce cell growth. The success of a structure for promotingcell growth depends fundamentally on the ability to continuously supplynutrients to the growing cells and to remove waste products that arecreated by the cell metabolism.

In accordance with embodiments of the present invention, microfluidicfree interface diffusion could be employed to continuously supply cellswith needed food, and to eliminate waste products. Cells could beintroduced and then contained in chambers that are connected to variousreservoirs containing food, growth factors, buffer, and other materialsto support cell growth, via channels that are too small to let the cellspass. Free interface diffusion could then be used to control the flux offood, growth factors, or waste to and from the chambers.

For example, FIGS. 27A and 27B show plan and cross-sectional views(along line 45B-45B′) respectively, of one embodiment of a cell cagestructure in accordance with the present invention. Cell cage 4500 isformed as an enlarged portion 4500 a of a flow channel 4501 in anelastomeric block 4503 in contact with substrate 4505. Cell cage 4500 issimilar to an individual cell pen as described above in FIGS. 26A-D,except that ends 4500 b and 4500 c of cell cage 4500 do not completelyenclose interior region 4500 a. Rather, ends 4500 a and 4500 b of cage4500 are formed by a plurality of retractable pillars 4502. Pillars 4502may be part of a membrane structure of a normally-closed valve structureas described extensively above in connection with FIGS. 21A-J.

Specifically, control channel 4504 overlies pillars 4502. When thepressure in control channel 4504 is reduced, elastomeric pillars 4502are drawn upward into control channel 4504, thereby opening end 4500 bof cell cage 4500 and permitting a cell to enter. Upon elevation ofpressure in control channel 4504, pillars 4502 relax downward againstsubstrate 4505 and prevent a cell from exiting cage 4500.

Elastomeric pillars 4502 are of a sufficient size and number to preventmovement of a cell out of cage 4500, but also include gaps 4508 whichallow the flow or diffusion of nutrients into cage interior 4500 a inorder to sustain cell(s) stored therein. Pillars 4502 on opposite end4500 c are similarly configured beneath second control channel 4506 topermit opening of the cage and removal of the cell as desired.

Microfluidic free interface diffusion in accordance with embodiments ofthe present invention could be utilized to provide a steady supply ofnutrients and growth factors to cells growing within the cell cage, andto remove waste products such as low molecular weight salts from thecell cage. One embodiment is shown in FIG. 48, wherein cell cage 8200 isin fluid communication with dead-ended branch channel 8202 of flowchannel 8204, which supplies nutrients 8205 by diffusion across themicrofluidic free interface 8206. Waste products 8207 from cell cage8200 in turn also diffuse across microfluidic free interface 8206 andenter into flow channel 8204 for removal.

Control over the rate of diffusion of materials to and from the cellcage could be accomplished by varying microfluidic channel dimensionssuch as width, height, and length, the number of connecting channels,the size of reservoirs, the size of chambers, and the concentration ofnutrient supplies and or waste products. By varying the above-listedfactors, amongst others, it is possible to regulate the rate of cellgrowth and division, and hence the maximum density of cells. And becauseat microfluidic dimensions there may be no appreciable concentrationgradient within larger volume chambers connected by a narrow channel,the entire cell population within such a chamber may be exposedsimultaneously to similar conditions.

Utilizing microfluidic free interface diffusion in accordance withembodiments of the present invention, it may also be possible to culturetwo or more different strains of cells positioned in adjacent chambers,while allowing for diffusion of nutrients, waste, and other products ofcell growth to move between the chambers. This type of microfluidicstructure and method may be particularly valuable in studyinginteractions between proximate cells, including but not limited to cellsignaling, cell/cell toxicity, and cell/cell symbiosis.

Finally, microfluidic free interface diffusion in accordance withembodiments of the present invention could further be used to establishgradients of nutrient/waste conditions in order to screen for desiredcell growth conditions. The use of diffusion for growing cell culturesis practical at these small dimensions, where diffusion lengths areshort and volumes are sufficiently small to allow diffusive transport tosignificantly change conditions over a short period of time.

10. Chemotaxis

Still another potential application for embodiments in accordance withthe present invention is the study of chemotaxis. Chemotaxis is thechange in direction of movement of a motile cell in response to aconcentration gradient of a specific chemical. This change in thedirection may result in the cell moving toward or away from the sensedchemical at a specific rate. Chemotaxis plays a key role inimmuno-responses, wherein components of the immune system migrate to anddestroy foreign agents in response to a sensed change in chemicalenvironment attributable to the presence of the foreign agents.

Microfluidic free interfaces created accordance with embodiments of thepresent invention may provide a valuable way to precisely reproducechemical concentration gradients. The activity of motile cells inresponse to exposure to these concentration gradients would enableresearchers to study and understand the phenomenon of chemotaxis.

11. Creating a Polymer with Cross-Linking/Porosity Gradient

In many biological applications, polymerized gels are used to separatemolecules based on their differing mobility through the gel, which istypically of uniform composition. However, the ability of the get toefficiently separate molecules may be improved by imposing a gradient onproperties such as pH, density, or polymerization cross-linking duringformation of the gel.

Thus in accordance with an embodiment of the present invention,microfluidic free interface diffusion may be used to fabricatepolymerized gels having well defined gradients of particular properties.For instance, in one example a concentrated, unpolymerized,photo-curable gel (such as agarose) may be introduced into amicrofluidic chamber in fluid communication via a microfluidic channelwith a second microfluidic chamber containing a buffer solution. As thecontents of the respective chambers diffuse into one another through thechannel, a gel concentration gradient is established. Once this gradienthas formed, the gel may be exposed to UV radiation to causecross-linking creating a solid gel. The gel concentration gradient, andhence the porosity of the gel, is fixed into the gel as a result of thispolymerization.

A gel exhibiting a gradient as just described may be used to determinethe size of a macromolecule by applying a potential and allowing themolecule to migrate through the gel from the area of greatest porosityand lowest gel concentration, to the area of least porosity and highestgel concentration. Once the average pore size of the gel becomes toosmall to allow the molecule to pass, the molecule will remain trapped inthe gel and detectable by standard fluorescent or silver stainingmethods. The location of the molecules in the gel would therefore pose adirect reflection of the size of the molecules.

In an alternative embodiment of forming gels with superimposed gradientsusing microfluidic free interface diffusion in accordance with thepresent invention, the polymerized gel may be formed with an imprintedpH gradient. In such an alternative embodiment, a first chamber isfilled with an unpolymerized gel with one buffer at one pH and a secondchamber is filled with a combination of the unpolymerized gel at thesame concentration with a second buffer at a second pH. The moleculesare then allowed to diffuse across the microfluidic channel connectingthe chambers to establish a pH gradient. The gel is then polymerizedcreating a solid gel of uniform porosity. Where the diffusing moleculeis capable of being permanently bonded to the gel matrix, a uniform pHgradient may be permanently established along the length of theconnecting channel. Such a pH gradient may be employed in combinationwith an applied electric field to separate molecules moving through thegel based upon their different isoelectric points.

12. Drug Delivery Systems

The ability to control the rate of flux out of or into a microfluidicchamber enables the timed release of chemicals or drugs, without theneed for active control strategies such as clocks. For example, animplanted microfluidic drug delivery device could contain a plurality ofchambers connected to an exit orifice through microfluidic channels ofvarying geometries. In this way the rate of transport of each of thechemicals in the chambers could be controlled separately and incombination. Utilizing such a structure, several different drugs couldbe introduced to the body at different and well defined rates.

For example, in an embodiment of a drug delivery device utilizingmicrofluidic free interface diffusion, a plurality of chambers may firstbe connected via channels of varying dimensions to chambers filled witha buffer solution. These buffer-filled chambers may then in turn beconnected via channels of various dimensions to an exit orifice.Dimensions of the microfluidic chambers and connecting channels wouldallow control over both the rate and timing of release of the drugs intothe body from the chambers. By judicious selection of channel dimensionssuch as width and length, and chamber volumes and shapes, and drugconcentrations, it is possible to achieve very precise control over thetotal rate of drug delivery over time.

13. Mixing of Highly Reactive Chemical Species

Still another example of a potential application for microfluidic freeinterfaces in accordance with embodiments of the present invention is inthe controlled mixing of highly reactive species. For example, undercertain circumstances it may be desirable to cause a reaction to occurat a slow or gradual pace between two or more chemical species. Underconventional conditions involving convective flow however, the speciesmay react suddenly and/or violently, mitigating the usefulness of thereaction products.

In accordance with embodiments of the present invention, however,contact and reaction between the chemical species is governedexclusively by diffusion with no convective flow. Therefore it may bepossible to conduct a reaction between the chemical species at a slowedand governable pace. One field wherein embodiments in accordance withthe present invention could offer a considerable advantage is in thepreparation of explosives or other potentially unstable products,wherein rapidly changing reactant concentrations could give rise tohazardous or otherwise undesirable conditions. Embodiments in accordancewith the present invention could also be potentially useful forcombinitoric synthesis applications where an end or intermediate productis unstable, highly reactive, and/or volatile.

14. Biological Assays

Embodiments of microfluidic fee interfaces in accordance with thepresent invention may be employed for a variety of applications.Examples of such applications are summarized below. A more completedescription of possible applications may be found in PCT applicationPCT/US01/44869, filed Nov. 16, 2001 and entitled “Cell Assays and HighThroughput Screening”, hereby incorporated by reference for allpurposes. Examples of microfluidic structures suitable for performingsuch applications include those described herein, as well as othersdescribed in U.S. patent application Ser. No. 10/118,466, “Nucleic AcidAmplification Utilizing Microfluidic Devices”, filed Apr. 5, 2002,hereby incorporated by reference for all purposes.

A wide variety of binding assays can be conducted utilizing themicrofluidic devices disclosed herein. Interactions between essentiallyany ligand and antiligand can be detected. Examples of ligand/antiligandbinding interactions that can be investigated include, but are notlimited to, enzyme/ligand interactions (e.g., substrates, cofactors,inhibitors); receptor/ligand; antigen/antibody; protein/protein(homophilic/heterophilic interactions); protein/nucleic acid; DNA/DNA;and DNA/RNA. Thus, the assays can be used to identify agonists andantagonists to receptors of interest, to identify ligands able to bindreceptors and trigger an intracellular signal cascade, and to identifycomplementary nucleic acids, for example. Assays can be conducted indirect binding formats in which a ligand and putative antiligand arecontacted with one another or in competitive binding formats well knownto those of ordinary skill in the art.

Heterogenous binding assays involve a step in which complexes areseparated from unreacted agents so that labeled complexes can bedistinguished from uncomplexed labeled reactants. Often this is achievedby attaching either the ligand or antiligand to a support. After ligandsand antiligands have been brought into contact, uncomplexed reactantsare washed away and the remaining complexes subsequently detected.

The binding assays conducted with the microfluidic devices providedherein can also be conducted in homogeneous formats. In the homogeneousformats, ligands and antiligands are contacted with one another insolution and binding complexes detected without having to removeuncomplexed ligands and antiligands. Two approaches frequently utilizedto conduct homogenous assays are fluorescence polarization (FP) and FRETassays.

The microfluidic devices can also be utilized in a competitive formatsto identify agents that inhibit the interaction between known bindingpartners. Such methods generally involve preparing a reaction mixturecontaining the binding partners under conditions and for a timesufficient to allow the binding partners to interact and form a complex.In order to test a compound for inhibitory activity, the reactionmixture is prepared in the presence (test reaction mixture) and absence(control reaction mixture) of the test compound. Formation of complexesbetween binding partners is then detected, typically by detecting alabel borne by one or both of the binding partners. The formation ofmore complexes in the control reaction then in the test reaction mixtureat a level that constitutes a statistically significant differenceindicates that the test compound interferes with the interaction betweenthe binding partners.

Immunological assays are one general category of assays that can beperformed with the microfluidic devices in accordance with embodimentsof the present invention. Certain assays are conducted to screen apopulation of antibodies for those that can specifically bind to aparticular antigen of interest. In such assays, a test antibody orpopulation of antibodies is contacted with the antigen. Typically, theantigen is attached to a solid support. Examples of immunological assaysinclude enzyme linked immunosorbent assays (ELISA) and competitiveassays as are known in the art.

Utilizing the microfluidic devices provided herein, a variety ofenzymatic assays can be performed. Such enzymatic assays generallyinvolve introducing an assay mixture containing the necessary componentsto conduct an assay into the various branch flow channels. The assaymixtures typically contain the substrate(s) for the enzyme, necessarycofactors (e.g., metal ions, NADH, NAPDH), and buffer, for example. If acoupled assay is to be performed, the assay solution will also generallycontain the enzyme, substrate(s) and cofactors necessary for theenzymatic couple.

Microfluidic devices in accordance with embodiments of the presentinvention can be arranged to include a material that selectively bindsto an enzymatic product that is produced. In some instances, thematerial has specific binding affinity for the reaction product itself.Somewhat more complicated systems can be developed for enzymes thatcatalyze transfer reactions. Certain assays of this type, for example,involve incubating an enzyme that catalyzes the transfer of a detectablemoiety from a donor substrate to an acceptor substrate that bears anaffinity label to produce a product bearing both the detectable moietyand the affinity label. This product can be captured by material thatincludes a complementary agent that specifically binds to the affinitylabel. This material typically is located in a detection region suchthat captured product can be readily detected. In certain assays, thematerial is coated to the interior channel walls of the detectionsection; alternatively, the material can be a support located in thedetection region that is coated with the agent.

Certain assays utilizing the present devices are conducted with vesiclesrather than cells. Once example of such an assay is a G-protein coupledreceptor assay utilizing fluorescent correlation spectroscopy (FCS).Membrane vesicles constructed from cells that over-express the receptorof interest are introduced into a main flow channel. Vesicles can eitherbe premixed with inhibitor and introduced via branch flow channels orvia one of the main flow channels prior to being mixed with afluorescent natural ligand which is also introduced by a main flowchannel. Components are allowed to incubate for the desired time andfluorescent signals may be analyzed directly in the flow chamber usingan FCS reader such as the Evotec/Zeiss Confocor (a single or dual photoncounting device).

FRET assays can also be utilized to conduct a number of ligand-receptorinteractions using the devices disclosed herein. For example, a FRETpeptide reporter can be constructed by introducing a linker sequence(corresponding to an inducible domain of a protein such as aphosphorylation site) into a vector encoding for a fluorescent proteincomposed of blue- and red-shifted GFP variants. The vector can be abacterial (for biochemical studies) or a mammalian expression vector(for in vivo studies).

Assays of nuclear receptors can also be performed with the presentmicrofluidic devices. For example, FRET-based assays forco-activator/nuclear receptor interaction can be performed. As aspecific example, such assays can be conducted to detect FRETinteractions between: (a) a ligand binding domain of a receptor taggedwith CFP (cyan fluorescent protein, a GFP derivative), and (b) areceptor binding protein (a coactivator) tagged with the Yellowfluorescent protein (YFP).

Fluorescence polarization (FP) can be utilized to develop highthroughput screening (HTS) assays for nuclear receptor-liganddisplacement and kinase inhibition. Because FP is a solution-based,homogeneous technique, there is no requirement for immobilization orseparation of reaction components. In general, the methods involve usingcompetition between a fluorescently labeled ligand for the receptor andrelated test compounds.

A number of different cell reporter assays can be conducted with theprovided microfluidic devices. One common type of reporter assay thatcan be conducted include those designed to identify agents that can bindto a cellular receptor and trigger the activation of an intracellularsignal or signal cascade that activates transcription of a reporterconstruct. Such assays are useful for identifying compounds that canactivate expression of a gene of interest. Two-hybrid assays, discussedbelow, are another major group of cell reporter assays that can beperformed with the devices. The two-hybrid assays are useful forinvestigating binding interactions between proteins.

Often cell reporter assays are utilized to screen libraries ofcompounds. In general such methods involve introducing the cells intothe main flow channel so that cells are retained in the chambers locatedat the intersection between the main flow channel and branch channels.Different test agents (e.g., from a library) can then be introduced intothe different branch channels where they become mixed with the cells inthe chambers. Alternatively, cells can be introduced via the main flowchannel and then transferred into the branch channel, where the cellsare stored in the holding areas. Meanwhile, different test compounds areintroduced into the different branch flow channels, usually to at leastpartially fill the chambers located at the intersection of the main andbranch flow channels. The cells retained in the holding area can bereleased by opening the appropriate valves and the cells transferred tothe chambers for interaction with the different test compounds. Once thecells and test compounds have been mixed, the resulting solution isreturned to the holding space or transported to the detection sectionfor detection of reporter expression. The cells and test agents canoptionally be further mixed and incubated using mixers of the design setforth above.

Cells utilized in screening compounds to identify those able to triggergene expression typically express a receptor of interest and harbor aheterologous reporter construct. The receptor is one which activatestranscription of a gene upon binding of a ligand to the receptor. Thereporter construct is usually a vector that includes a transcriptionalcontrol element and a reporter gene operably linked thereto. Thetranscriptional control element is a genetic element that is responsiveto an intracellular signal (e.g., a transcription factor) generated uponbinding of a ligand to the receptor under investigation. The reportergene encodes a detectable transcriptional or translational product.Often the reporter (e.g., an enzyme) can generate an optical signal thatcan be detected by a detector associated with a microfluidic device.

A wide variety of receptor types can be screened. The receptors oftenare cell-surface receptors, but intracellular receptors can also beinvestigated provided the test compounds being screened are able toenter into the cell. Examples of receptors that can be investigatedinclude, but are not limited to, ion channels (e.g., calcium, sodium,potassium channels), voltage-gated ion channels, ligand-gated ionchannels (e.g., acetyl choline receptors, and GABA (gamma-aminobutyricacid) receptors), growth factor receptors, muscarinic receptors,glutamate receptors, adrenergic receptors, dopamine receptors.

Another general category of cell assays that can be performed is the twohybrid assays. In general, the two-hybrid assays exploit the fact thatmany eukaryotic transcription factors include a distinct DNA-bindingdomain and a distinct transcriptional activation domain to detectinteractions between two different hybrid or fusion proteins. Thus, thecells utilized in two-hybrid assays include the construct(s) that encodefor the two fusion proteins. These two domains are fused to separatebinding proteins potentially capable of interacting with one anotherunder certain conditions. The cells utilized in conducting two-hybridassays contain a reporter gene whose expression depends upon either aninteraction, or lack of interaction, between the two fusion proteins.

In addition to the assays just described, a variety of methods to assayfor cell membrane potential can be conducted with the microfluidicdevices disclosed herein. In general, methods for monitoring membranepotential and ion channel activity can be measured using two alternatemethods. One general approach is to use fluorescent ion shelters tomeasure bulk changes in ion concentrations inside cells. The secondgeneral approach is to use of FRET dyes sensitive to membrane potential.

The microfluidic devices disclosed herein can be utilized to conduct avariety of different assays to monitor cell proliferation. Such assayscan be utilized in a variety of different studies. For example, the cellproliferation assays can be utilized in toxicological analyses, forexample. Cell proliferation assays also have value in screeningcompounds for the treatment of various cell proliferation disordersincluding tumors.

The microfluidic devices disclosed herein can be utilized to perform avariety of different assays designed to identify toxic conditions,screen agents for potential toxicity, investigate cellular responses totoxic insults and assay for cell death. A variety of differentparameters can be monitored to assess toxicity. Examples of suchparameters include, but are not limited to, cell proliferation,monitoring activation of cellular pathways for toxicological responsesby gene or protein expression analysis, DNA fragmentation; changes inthe composition of cellular membranes, membrane permeability, activationof components of death-receptors or downstream signaling pathways (e.g.,caspases), generic stress responses, NF-kappaB activation and responsesto mitogens. Related assays are used to assay for apoptosis (aprogrammed process of cell death) and necrosis.

By contacting various microbial cells with different test compounds, onecan also utilize the devices provided herein to conduct antimicrobialassays, thereby identifying potential antibacterial compounds. The term“microbe” as used herein refers to any microscopic and/or unicellularfungus, any bacteria or any protozoan. Some antimicrobial assays involveretaining a cell in a cell cage and contacting it with at least onepotential antimicrobial compound. The effect of the compound can bedetected as any detectable change in the health and/or metabolism of thecell. Examples of such changes, include but are not limited to,alteration in growth, cell proliferation, cell differentiation, geneexpression, cell division and the like.

Certain of the microfluidic devices provided herein can be utilized toconduct mini-sequencing reactions or primer extension reactions toidentify the nucleotide present at a polymorphic site in a targetnucleic acid. In general, in these methods a primer complementary to asegment of a target nucleic acid is extended if the reaction isconducted in the presence of a nucleotide that is complementary to thenucleotide at the polymorphic site. Often such methods are single basepair extension (SBPE) reactions. Such method typically involvehybridizing a primer to a complementary target nucleic acid such thatthe 3′ end of the primer is immediately adjacent the polymorphic site,or is a few bases upstream of the polymorphic site. The extensionreaction is conducted in the presence of one or more labelednon-extendible nucleotides (e.g., dideoxynucleotides) and a polymerase.Incorporation of a non-extendible nucleotide onto the 3′ end of theprimer prevents further extension of the primer by the polymerase oncethe non-extendible nucleotide is incorporated onto the 3′ end of theprimer.

Related to the methods just described, the present devices can also beutilized to amplify and subsequently identify target nucleic acids inmultiple samples using amplification techniques that are wellestablished in the art. In general such methods involve contacting asample potentially containing a target nucleic acid with forward andreverse primers that specifically hybridize to the target nucleic acid.The reaction includes all four dNTPs and polymerase to extend the primersequences.

While the present invention has been described herein with reference toparticular embodiments thereof, a latitude of modification, variouschanges and substitutions are intended in the foregoing disclosure, andit will be appreciated that in some instances some features of theinvention will be employed without a corresponding use of other featureswithout departing from the scope of the invention as set forth.Therefore, many modifications may be made to adapt a particularsituation or material to the teachings of the invention withoutdeparting from the essential scope and spirit of the present invention.It is intended that the invention not be limited to the particularembodiment disclosed as the best mode contemplated for carrying out thisinvention, but that the invention will include all embodiments andequivalents falling within the scope of the claims.

1-41. (canceled)
 42. A method of creating a concentration gradient of achemical species comprising: disposing a first fluid containing thechemical species; disposing a second static fluid proximate to the firststatic fluid to form a microfluidic free interface; suppressingconvective flow of the first and second fluids such that mixing betweenthe first and second fluids across the microfluidic free interfaceoccurs substantially exclusively by diffusion and a concentrationgradient of the chemical species is created.
 43. The method of claim 42wherein the first chemical species comprises a nutrient for a cell influidic communication with the second fluid.
 44. The method of claim 42further comprising: disposing a third fluid containing a second chemicalspecies proximate to the second fluid to form a second microfluidic freeinterface; suppressing convective flow of the second and third fluidssuch that mixing between the second and third fluids across the secondmicrofluidic free interface occurs substantially exclusively bydiffusion and a second concentration gradient of the second chemicalspecies is superimposed over the concentration gradient of the firstchemical species.
 45. The method of claim 44 wherein a direction of theconcentration gradient of the first chemical species is not parallelwith a direction of the concentration gradient of the second chemicalspecies.
 46. The method of claim 44 wherein the direction of theconcentration gradient of the first chemical is approximatelyperpendicular to a direction of the concentration gradient of the secondchemical, such that an array of concentration conditions of the firstand second chemical species is created.
 47. The method of claim 46wherein diffusion of the first and second chemical species occurs in ashallow chamber.
 48. The method of claim 46 wherein diffusion of thefirst and second chemical species occurs in a set oforthogonally-oriented microfluidic flow channels. 49-51. (canceled)